TWI714022B - Anti-pdl1 antibody formulations - Google Patents

Anti-pdl1 antibody formulations Download PDF

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TWI714022B
TWI714022B TW108108366A TW108108366A TWI714022B TW I714022 B TWI714022 B TW I714022B TW 108108366 A TW108108366 A TW 108108366A TW 108108366 A TW108108366 A TW 108108366A TW I714022 B TWI714022 B TW I714022B
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antibody
cancer
formulation
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TW201936209A (en
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瑩 楊
史瑞達拉 愛拉維坦
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美商建南德克公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Abstract

The invention provides stable aqueous pharmaceutical formulations comprising an anti-PDL1 antibody. The invention also provides methods for making such formulations and methods of using such formulations.

Description

抗-PDL1抗體調配物Anti-PDL1 antibody formulation

本發明係關於穩定水性醫藥調配物，其包含抗PDL1抗體。The present invention relates to a stable aqueous pharmaceutical formulation comprising an anti-PDL1 antibody.

向T細胞提供兩種不同信號為廣泛接受的藉由抗原呈現細胞(APC)進行休眠T淋巴細胞之淋巴細胞活化的模型。Lafferty等人，Aust. J. Exp. Biol. Med. ScL 53:27-42 (1975)。此模型進一步提供區別自身與非自身之辨別力及免疫耐受性。Bretscher等人，Science 169: 1042-1049 (1970)；Bretscher, P.A., P.N.A.S. USA 96: 185-190 (1999)；Jenkins等人，J. Exp. Med. 165:302-319 (1987)。在識別在主要組織相容性複合物(MHC)之情形下存在的外來抗原肽之後，第一信號或抗原特異性信號經T細胞受體(TCR)轉導。第二信號或協同刺激信號藉由表現於抗原呈現細胞(APC)上之協同刺激分子傳遞至T細胞，且誘導T細胞促進純系擴增、細胞激素分泌及效應功能。Lenschow等人，Ann. Rev. Immunol. 14:233 (1996)。在無協同刺激存在下，T細胞會變得對抗原刺激無反應，不產生有效免疫反應且可能進一步導致外來抗原耗竭或耐受外來抗原。 在兩信號模型中，T細胞接收正性第二協同刺激信號及負性第二協同刺激信號。該等正性與負性信號之調控對宿主之保護性免疫反應最大化至關重要，同時維持免疫耐受性及防止產生自體免疫。負性第二信號對誘導T細胞耐受性而言似乎必不可少，而正性信號促進T細胞活化。儘管簡單的雙信號模型仍能有效說明原生淋巴細胞，但宿主之免疫反應為動力學過程，且亦可向暴露於抗原中之T細胞提供協同刺激信號。協同刺激之機制具有治療意義，此係因為協同刺激信號之處理已展示提供一種增進或終止基於細胞之免疫反應之方法。近來，已發現T細胞功能障礙或因應性缺失(anergy)與抑制受體(漸進式死亡多肽1(PD-1))之經誘導及維持表現同時出現。因此，靶向PD-1及通過與PD-1相互作用發信號之其他分子(諸如程式化死亡配位體1 (PD-Ll)及程式化死亡配位體2 (PD-L2))之治療為密切關注之領域。 PD-L1在許多癌症中過度表現且通常預後不佳(Okazaki T等人，Intern. Immun. 2007 19(7):813) (Thompson RH等人，Cancer Res 2006, 66(7):3381)。有趣的是，與正常組織中之T淋巴細胞及周邊血液T淋巴細胞相對，大部分浸潤T淋巴細胞之腫瘤主要表現PD-1，表明腫瘤反應性T細胞上PD-1之上調可造成抗腫瘤免疫反應減弱(Blood 2009 114(8):1537)。此可歸因於使用由表現PD-L1之腫瘤細胞介導之PD-L1信號傳導，該等腫瘤細胞與表現PD-1之T細胞相互作用，導致T細胞活化減弱及逃避免疫監視(Sharpe等人，Nat Rev 2002)(Keir ME等人，2008 Annu. Rev. Immunol. 26:677)。因此，抑制PD-L1/PD-1相互相用可促進CD8+T細胞介導之腫瘤殺死。 靶向PD-1及通過與PD-1相互作用發信號之其他分子(諸如程式化死亡配位體1 (PD-Ll)及程式化死亡配位體2 (PD-L2))之治療為密切關注之領域。已提出抑制PD-L1信號傳導作為增進T細胞免疫以治療癌症(例如腫瘤免疫)及感染(包括急性與慢性(例如持續性)感染)之方法。然而，因為針對此路徑中之目標之最佳治療法尚未商業化，所以存在明顯未滿足之醫療需求。 本文所引用之所有參考文獻(包括專利申請案、專利公開案及UniProtKB/Swiss-Prot寄存編號)均以全文引用之方式併入本文中，就像各個別參考文獻特定地且個別地以引用之方式併入一般。Providing two different signals to T cells is a widely accepted model of lymphocyte activation of resting T lymphocytes by antigen presenting cells (APC). Lafferty et al., Aust. J. Exp. Biol. Med. ScL 53:27-42 (1975). This model further provides discrimination and immune tolerance to distinguish self from non-self. Bretscher et al., Science 169: 1042-1049 (1970); Bretscher, P.A., P.N.A.S. USA 96: 185-190 (1999); Jenkins et al., J. Exp. Med. 165:302-319 (1987). After recognizing the foreign antigen peptide present in the context of the major histocompatibility complex (MHC), the first signal or antigen-specific signal is transduced via the T cell receptor (TCR). The second signal or costimulatory signal is delivered to T cells by costimulatory molecules expressed on antigen-presenting cells (APC), and induces T cells to promote clone expansion, cytokine secretion, and effector functions. Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In the absence of co-stimulation, T cells will become unresponsive to antigen stimulation, do not produce an effective immune response and may further lead to foreign antigen exhaustion or tolerance to foreign antigens. In the two-signal model, T cells receive a positive second co-stimulatory signal and a negative second co-stimulatory signal. The regulation of these positive and negative signals is essential to maximize the host's protective immune response, while maintaining immune tolerance and preventing autoimmunity. Negative second signals seem to be essential for inducing T cell tolerance, while positive signals promote T cell activation. Although a simple dual-signal model can still effectively explain native lymphocytes, the host's immune response is a dynamic process and can also provide co-stimulatory signals to T cells exposed to antigens. The mechanism of co-stimulation has therapeutic significance because the processing of co-stimulatory signals has been shown to provide a method to enhance or stop the cell-based immune response. Recently, it has been found that T cell dysfunction or anergy and the induced and maintained performance of the inhibitory receptor (progressive death polypeptide 1 (PD-1)) occur simultaneously. Therefore, treatments that target PD-1 and other molecules that signal by interacting with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) It is an area of close attention. PD-L1 is overexpressed in many cancers and usually has a poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, compared with T lymphocytes in normal tissues and peripheral blood T lymphocytes, most tumors infiltrating T lymphocytes mainly show PD-1, indicating that upregulation of PD-1 on tumor-reactive T cells can cause anti-tumor The immune response is weakened (Blood 2009 114(8):1537). This can be attributed to the use of PD-L1 signal transduction mediated by tumor cells expressing PD-L1. These tumor cells interact with T cells expressing PD-1, resulting in weakened T cell activation and evasion of immune surveillance (Sharpe et al. Human, Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of PD-L1/PD-1 interaction can promote CD8+T cell-mediated tumor killing. Targeting PD-1 and other molecules that signal through interaction with PD-1 (such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2)) are closely related Area of concern. Inhibition of PD-L1 signaling has been proposed as a method to enhance T cell immunity to treat cancer (eg tumor immunity) and infections (including acute and chronic (eg persistent) infections). However, because the best treatment for the goals in this path has not been commercialized, there is a clear unmet medical need. All references cited in this article (including patent applications, patent publications and UniProtKB/Swiss-Prot deposit numbers) are incorporated into this article by reference in their entirety, just as each individual reference is specifically and individually cited The way is merged into the general.

相關申請案之交叉引用 本申請案主張2013年9月27日提交之美國臨時申請案第61/883,953號之優先權，該案以全文引用之方式併入本文中。 本文提供穩定水性醫藥調配物，其包含抗體。該調配物包含抗體(例如抗PDL1抗體)、緩衝液、蔗糖及界面活性劑，其中調配物之pH為約5.0至約7.0。 在一個態樣中，本文提供一種穩定水性醫藥調配物，該調配物包含濃度為約40 mg/ml至約125 mg/ml之抗PDL1單株抗體，濃度為約15 mM至約25 mM之組胺酸乙酸鹽或乙酸鈉，濃度為約60 mM至約240 mM之蔗糖，濃度為約0.005% (w/v)至約0.06% (w/v)之聚山梨醇酯，且pH為約5.0至約6.3。 在一些實施例中，調配物中之單株抗體為約40 mg/ml至約80 mg/ml。在一些實施例中，調配物中之單株抗體為約54 mg/ml至約66 mg/ml。在一些實施例中，調配物中之單株抗體為約60 mg/ml。在一些實施例中，調配物中之單株抗體為約60 mg/ml至約125 mg/ml。在一些實施例中，調配物中之單株抗體為約125 mg/ml。 在一些實施例中，調配物中之該組胺酸乙酸鹽或乙酸鈉之濃度為約17 mM至約22 mM。在一些實施例中，調配物中之該組胺酸乙酸鹽或乙酸鈉之濃度為約20 mM。 在一些實施例中，調配物中之該蔗糖為約60 mM至約180 mM。在一些實施例中，調配物中之該蔗糖為約120 mM。在一些實施例中，調配物中之該蔗糖為約240 mM。 在一些實施例中，調配物之pH為約5.5至約6.1。在一些實施例中調配物之pH為約5.5或約5.8。 在一些實施例中，調配物中之該聚山梨醇酯為聚山梨醇酯20。在一些實施例中，調配物中之該聚山梨醇酯(例如聚山梨醇酯20)為約0.02%至約0.04%。 在一些實施例中，調配物中之該單株抗體為約60 mg/ml，調配物中之蔗糖為約120 mM，且pH為約5.8。在一些實施例中，調配物中之該單株抗體為約125 mg/ml，調配物中之蔗糖為約240 mM，且pH為約5.5。 在一些實施例中，調配物包含約60 mg/mL之量的單株抗體(例如本文所述之抗PDL1抗體)，約20 mM之濃度的組胺酸乙酸鹽，約120 mM之濃度的蔗糖及0.04% (w/v)之濃度的為聚山梨醇酯20之聚山梨醇酯，且調配物之pH為約5.8。 在一些實施例中，調配物包含約125 mg/mL之量的單株抗體，約20 mM之濃度的組胺酸乙酸鹽，約240 mM之濃度的蔗糖，及濃度為0.02%的為聚山梨醇酯20之聚山梨醇酯，且調配物之pH為約5.5。 在一些實施例中，調配物中之該單株抗體未進行先前凍乾。在一些實施例中，調配物中之該單株抗體為全長抗體。在一些實施例中，調配物中之該單株抗體為IgG1抗體。在一些實施例中，調配物中之該單株抗體為人類化抗體。在一些實施例中，調配物中之該單株抗體為包含抗原結合區之抗體片段。在一些實施例中，抗體片段為Fab或F(ab')2 片段。 在一些實施例中，調配物中之該單株抗體包含 (a)輕鏈可變區，其包含： (1) HVR-L1，其包含胺基酸序列RASQDVSTAVA (SEQ ID NO:1)； (2) HVR-L2，其包含胺基酸序列SASFLYS (SEQ ID NO:2)； (3) HVR-L3，其包含胺基酸序列QQYLYHPAT (SEQ ID NO:3)；及 (b)重鏈可變區，其包含： (1) HVR-H1，其包含胺基酸序列GFTFSDSWIH (SEQ ID NO:4)； (2) HVR-H2，其包含胺基酸序列AWISPYGGSTYYADSVKG (SEQ ID NO:5)； (3) HVR-H3，其包含胺基酸序列RHWPGGFDY (SEQ ID NO:6)。 在一些實施例中，調配物中之該單株抗體包含含有胺基酸序列SEQ ID NO:7之輕鏈可變區，及含有胺基酸序列SEQ ID NO:8之重鏈可變區。在一些實施例中，調配物中之該單株抗體包含與具有胺基酸序列SEQ ID NO:7之輕鏈可變區具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性的輕鏈可變區，及與具有胺基酸序列SEQ ID NO:8之重鏈可變區具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性的重鏈可變區。在一些實施例中，調配物中之該單株抗體包含含有胺基酸序列SEQ ID NO:7之輕鏈可變區，及含有胺基酸序列SEQ ID NO:32之重鏈可變區。在一些實施例中，調配物中之該單株抗體包含與具有胺基酸序列SEQ ID NO:7之輕鏈可變區具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性的輕鏈可變區，及與具有胺基酸序列SEQ ID NO:32之重鏈可變區具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性的重鏈可變區。在一些實施例中，調配物中之該單株抗體包含含有胺基酸序列SEQ ID NO:9之輕鏈及含有胺基酸序列SEQ ID NO:10之重鏈。在一些實施例中，調配物中之該單株抗體包含與具有胺基酸序列SEQ ID NO:9之輕鏈具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性之輕鏈，及與具有胺基酸序列SEQ ID NO:10之重鏈具有至少85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%一致性的重鏈。 在一些實施例中，包含抗體之調配物儲存於玻璃瓶或金屬合金容器中。在一些實施例中，金屬合金為316L不鏽鋼或赫史特合金(hastelloy)。在一些實施例中，調配物在2-8℃下穩定至少6個月、至少12個月、至少18個月或至少24個月。在一些實施例中，調配物中之抗體在儲存後保留至少約75%、至少約80%、至少約85%、至少約90%儲存前的生物活性。在一些實施例中，藉由結合於PD-L1之抗體量測生物活性。 在一些實施例中，本文所述之調配物為無菌的。在一些實施例中，本文所述之調配物適於向個體投與。在一些實施例中，本文所述之調配物用於靜脈內(IV)投與。 在另一態樣中，本文提供一種製品或套組，其包含容納上文及此處描述之任何穩定水性醫藥調配物的容器。在一些實施例中，容器為玻璃瓶或金屬合金容器。在一些實施例中，金屬合金為316L不鏽鋼或赫史特合金。 在另一態樣中，本文提供一種治療個體中之疾病或病症之方法，其包含向該個體投與有效量之本文所述之調配物，其中該疾病或病症係選自由感染、癌症及發炎疾病組成之群。 應理解，可組合本文所述之各種實施例的一個、一些或所有特性形成本發明之其他實施例。本發明之此等及其他態樣對熟習此項技術者變得顯而易知。本發明之此等及其他實施例藉由下文之[實施方式]進一步描述。 Cross-reference of related applications This application claims the priority of U.S. Provisional Application No. 61/883,953 filed on September 27, 2013, which is incorporated herein by reference in its entirety. Provided herein is a stable aqueous pharmaceutical formulation comprising an antibody. The formulation includes an antibody (such as an anti-PDL1 antibody), buffer, sucrose, and a surfactant, wherein the pH of the formulation is about 5.0 to about 7.0. In one aspect, provided herein is a stable aqueous pharmaceutical formulation, the formulation comprising an anti-PDL1 monoclonal antibody at a concentration of about 40 mg/ml to about 125 mg/ml, and a concentration of about 15 mM to about 25 mM. Amino acid acetate or sodium acetate, sucrose with a concentration of about 60 mM to about 240 mM, polysorbate with a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 5.0 To about 6.3. In some embodiments, the monoclonal antibody in the formulation is about 40 mg/ml to about 80 mg/ml. In some embodiments, the monoclonal antibody in the formulation is about 54 mg/ml to about 66 mg/ml. In some embodiments, the monoclonal antibody in the formulation is about 60 mg/ml. In some embodiments, the monoclonal antibody in the formulation is about 60 mg/ml to about 125 mg/ml. In some embodiments, the monoclonal antibody in the formulation is about 125 mg/ml. In some embodiments, the concentration of the histidine acetate or sodium acetate in the formulation is about 17 mM to about 22 mM. In some embodiments, the concentration of the histidine acetate or sodium acetate in the formulation is about 20 mM. In some embodiments, the sucrose in the formulation is about 60 mM to about 180 mM. In some embodiments, the sucrose in the formulation is about 120 mM. In some embodiments, the sucrose in the formulation is about 240 mM. In some embodiments, the pH of the formulation is about 5.5 to about 6.1. In some embodiments the pH of the formulation is about 5.5 or about 5.8. In some embodiments, the polysorbate in the formulation is polysorbate 20. In some embodiments, the polysorbate (such as polysorbate 20) in the formulation is about 0.02% to about 0.04%. In some embodiments, the monoclonal antibody in the formulation is about 60 mg/ml, the sucrose in the formulation is about 120 mM, and the pH is about 5.8. In some embodiments, the monoclonal antibody in the formulation is about 125 mg/ml, the sucrose in the formulation is about 240 mM, and the pH is about 5.5. In some embodiments, the formulation contains a monoclonal antibody (such as the anti-PDL1 antibody described herein) in an amount of about 60 mg/mL, histidine acetate at a concentration of about 20 mM, and sucrose at a concentration of about 120 mM. And the concentration of 0.04% (w/v) is polysorbate of polysorbate 20, and the pH of the formulation is about 5.8. In some embodiments, the formulation contains monoclonal antibodies in an amount of about 125 mg/mL, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 240 mM, and polysorbate at a concentration of 0.02%. The polysorbate of alcohol ester 20, and the pH of the formulation is about 5.5. In some embodiments, the monoclonal antibody in the formulation has not been previously lyophilized. In some embodiments, the monoclonal antibody in the formulation is a full-length antibody. In some embodiments, the monoclonal antibody in the formulation is an IgG1 antibody. In some embodiments, the monoclonal antibody in the formulation is a humanized antibody. In some embodiments, the monoclonal antibody in the formulation is an antibody fragment comprising an antigen binding region. In some embodiments, the antibody fragment is a Fab or F(ab') 2 fragment. In some embodiments, the monoclonal antibody in the formulation comprises (a) a light chain variable region, which comprises: (1) HVR-L1, which comprises the amino acid sequence RASQDVSTAVA (SEQ ID NO:1); ( 2) HVR-L2, which includes the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3, which includes the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) the heavy chain can A variable region, which comprises: (1) HVR-H1, which comprises the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2, which comprises the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3, which contains the amino acid sequence RHWPGGFDY (SEQ ID NO: 6). In some embodiments, the monoclonal antibody in the formulation comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the monoclonal antibody in the formulation comprises at least 85%, 86%, 87%, 88%, 89%, 90%, and the light chain variable region having the amino acid sequence SEQ ID NO: 7 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the light chain variable region, and with the amino acid sequence SEQ ID NO: 8 The heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical heavy chain variable region. In some embodiments, the monoclonal antibody in the formulation comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 32. In some embodiments, the monoclonal antibody in the formulation comprises at least 85%, 86%, 87%, 88%, 89%, 90%, and the light chain variable region having the amino acid sequence SEQ ID NO: 7 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the light chain variable region, and with the amino acid sequence SEQ ID NO: 32 The heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical heavy chain variable region. In some embodiments, the monoclonal antibody in the formulation comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the monoclonal antibody in the formulation comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91% and the light chain having the amino acid sequence of SEQ ID NO: 9 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the light chain, and the heavy chain with the amino acid sequence SEQ ID NO: 10 has at least 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% consistent heavy chain. In some embodiments, the antibody-containing formulation is stored in a glass bottle or metal alloy container. In some embodiments, the metal alloy is 316L stainless steel or Hastelloy. In some embodiments, the formulation is stable at 2-8°C for at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the antibody in the formulation retains at least about 75%, at least about 80%, at least about 85%, at least about 90% of the biological activity before storage after storage. In some embodiments, the biological activity is measured by antibodies that bind to PD-L1. In some embodiments, the formulations described herein are sterile. In some embodiments, the formulations described herein are suitable for administration to an individual. In some embodiments, the formulations described herein are for intravenous (IV) administration. In another aspect, provided herein is an article or kit comprising a container containing any of the stable aqueous pharmaceutical formulations described above and herein. In some embodiments, the container is a glass bottle or a metal alloy container. In some embodiments, the metal alloy is 316L stainless steel or Hester alloy. In another aspect, provided herein is a method of treating a disease or disorder in an individual, which comprises administering to the individual an effective amount of the formulation described herein, wherein the disease or disorder is selected from infection, cancer, and inflammation A group of diseases. It should be understood that one, some or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to those familiar with the art. These and other embodiments of the present invention are further described by the following [Embodiments].

I. 定義 . 在詳細描述本發明之前，應理解本發明不限於特定組合物或生物系統，其當然可變化。亦應理解本文中使用之術語僅為描述特定實施例之目的而並非意欲具有限制性。如本說明書及隨附申請專利範圍中所使用，除非內容明確指出相反，否則單數形式「一」及「該」包括複數個指示物。因此，舉例而言，提及「分子」視情況包括兩個或兩個以上此類分子之組合，及其類似物。 如本文所用之術語「約」係指熟習此技術領域者容易知曉的各別值之常見誤差範圍。本文中提及「約」一值或參數包括(及描述)針對彼值或參數本身之實施例。 應理解，本文中所描述的本發明之態樣及實施例包括「包含」態樣及實施例、「由態樣及實施例組成」及「基本上由態樣及實施例組成」。 術語「醫藥調配物」係指呈使活性成份之生物活性有效之形式的製劑，且其不含對將投與該調配物之個體具有不可接受之毒性的額外組份。該等調配物為無菌的。「醫藥學上可接受之」賦形劑(媒劑、添加劑)為可合理投與個體哺乳動物以提供有效劑量之所用活性成份者。 「無菌」調配物為無菌的或不含或基本上不含所有活微生物及其孢子。 「冷凍」調配物為溫度在0℃以下之調配物。一般而言，冷凍調配物未經冷凍乾燥，亦未經受預先或後續凍乾。在某些實施例中，冷凍調配物包含便於儲存之冷凍原料藥(在不鏽鋼槽中)或冷凍藥品(最終小瓶組態)。 「穩定」調配物為在儲存後所含蛋白質基本上保持其物理穩定性及/或化學穩定性及/或生物活性的調配物。調配物在儲存後較佳基本上保持其物理及化學穩定性以及其生物活性。儲存期一般基於調配物之預期存放期選擇。此項技術中可獲得用於量測蛋白質穩定性之多種分析技術且，例如綜述於Peptide and Protein Drug Delivery , 247-301, Vincent Lee編, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)及Jones, A.Adv. Drug Delivery Rev. 10: 29-90 (1993)中。穩定性可在所選溫度下持續所選時段來量測。穩定性可以多種不同方式定性及/或定量評估，包括評估聚集物形成(例如使用尺寸排阻層析，藉由量測濁度及/或藉由目測)；藉由使用陽離子交換層析、成像毛細管等電聚焦(icIEF)或毛細管區帶電泳評定；胺基端或羧基端序列分析；質譜分析；比較縮短抗體與完整抗體之SDS-PAGE分析；肽圖譜(例如胰蛋白酶或LYS-C)分析；評估抗體之生物活性或抗原結合功能；等。不穩定性可涉及以下中任何一或多者：聚集、脫醯胺(例如Asn脫醯胺)、氧化(例如Met氧化)、異構化(例如Asp異構化)、剪接/水解/片段化(例如鉸鏈區片段化)、丁二醯亞胺形成、不成對半胱胺酸、N端擴展、C端加工、糖基化差異等。 若在目測檢查顏色及/或透明度或藉由UV光散射或藉由尺寸排阻層析量測時蛋白質展示無或極少聚集、沈澱及/或變性跡象，則在醫藥調配物中之蛋白質「保持其物理穩定性」。 若在既定時間下化學穩定性使得蛋白質被視為仍保持其如下所定義之生物活性，則在醫藥調配物中的蛋白質「保持其化學穩定性」。化學穩定性可藉由偵測及定量化學上變更形式之蛋白質來評估。化學變更可涉及尺寸修改(例如剪接)，其可例如使用尺寸排阻層析、SDS-PAGE及/或基質輔助雷射脫附離子化/飛行時間質譜分析(MALDI/TOF MS)來評估。其他類型之化學變更包括電荷變更(例如由脫醯胺所致)，其可藉由例如離子交換層析或icIEF來評估。 若如分析法(例如抗原結合分析)中所測定，既定時間之抗體的生物活性為醫藥調配物製備時展現之生物活性的至少約60%(在分析誤差內)，則醫藥調配物中之抗體「保留其生物活性」。在下文中詳述抗體之其他「生物活性」分析。 如本文所用，單株抗體之「生物活性」包括抗體結合於抗原且導致可活體外或活體內量測之可量測生物反應的能力。 本文中之「脫醯胺」單株抗體為一或多個天冬醯胺殘基已衍生為例如天冬胺酸或異天冬胺酸之單株抗體。 本文中之「氧化」單株抗體為一或多個色胺酸殘基及/或一或多個甲硫胺酸已經氧化之單株抗體。 本文中之「糖基化」單株抗體為一或多個離胺酸殘基已經糖基化之單株抗體。 「容易脫醯胺」之抗體為包含一或多個已發現有脫醯胺傾向之殘基的抗體。 「容易氧化」之抗體為包含一或多個已發現有氧化傾向之殘基的抗體。 「容易聚集」之抗體為已發現尤其在冷凍及/或攪拌之後與其他抗體分子聚集的抗體。 「容易片段化」之抗體為已發現在例如鉸鏈區裂解成兩個或兩個以上片段的抗體。 預期藉由「減少脫醯胺、氧化、聚集或片段化」來防止或降低關於在不同調配物中調配之單株抗體脫醯胺、氧化、聚集或片段化之量。 經調配之抗體較佳為基本上純的且希望基本上均勻(例如不含污染蛋白質等)。「基本上純的」抗體意謂組合物包含以組合物中之蛋白質總重量計至少約90重量%抗體，較佳至少約95重量%抗體。「基本上均質」抗體意謂組合物包含以組合物中之蛋白質總重量計至少約99重量%抗體。 「等張」意謂所關注調配物具有與人類血液基本上相同之滲透壓。等張調配物一般具有約250至350 mOsm之滲透壓。等張性可使用例如蒸汽壓或冰凍型滲壓計來量測。 如本文所用，「緩衝液」係指藉由酸-鹼共軛組份之作用阻止pH改變之緩衝溶液。本發明之緩衝液較佳具有約4.5至約7.0，較佳約5.6至約7.0，例如5.6至6.9、5.7至6.8、5.8至6.7、5.9至6.6、5.9至6.5、6.0、6.0至6.4或6.1至6.3範圍內之pH。在一個實施例中，緩衝液具有pH 5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9或7.0。舉例而言，磷酸鈉為將控制pH在此範圍中之緩衝液實例。 如本文所用，「界面活性劑」係指表面活性劑，較佳地非離子界面活性劑。本文中界面活性劑之實例包括聚山梨醇酯(例如聚山梨醇酯20及聚山梨醇酯80)；泊洛沙姆(poloxamer) (例如泊洛沙姆188)；曲拉通(Triton)；十二烷基硫酸鈉(SDS); 月桂基硫酸鈉；辛基醣苷鈉；月桂基-、肉豆蔻基-、亞油醇基-或硬脂醯基-磺基甜菜鹼；月桂基-、肉豆蔻基-、亞油醇基-或硬脂醯基-肌胺酸；亞油醇基-、肉豆蔻基-或鯨蠟基-甜菜鹼；月桂醯胺基丙基-、椰油醯胺基丙基-、亞油醯胺基丙基-、肉豆蔻醯胺基丙基-、棕櫚醯胺基丙基-或異硬脂醯胺基丙基-甜菜鹼(例如月桂醯胺基丙基)；肉豆蔻醯胺基丙基-、棕櫚醯胺丙基-或異硬脂醯胺基丙基-二甲胺；甲基椰油醯基牛磺酸鈉或甲基油醇牛磺酸二鈉；及MONAQUATTM 系列(Mona Industries, Inc., Paterson, N.J.)；聚乙二醇、聚丙二醇及乙二醇與丙二醇之共聚物(例如Pluronics, PF68等)；等。在一個實施例中，本文之界面活性劑為聚山梨醇酯20。 自藥理學角度來看，在本發明之上下文中，抗體之「治療有效量」係指有效預防或治療病症之量，其中抗體在該病症之治療中有效。「病症」為受益於使用抗體治療之任何病狀。此包括慢性及急性病症或疾病，包括使哺乳動物易患所論述病症之彼等病理學條件。 「防腐劑」為可視情況包括於調配物中以基本上減少其中之細菌作用，從而便於例如多用途調配物之製造的化合物。潛在防腐劑之實例包括氯化十八烷基二甲基苯甲基銨、氯化六羥季銨、氯化苯甲烴銨(烷基為長鏈化合物之氯化烷基苯甲基二甲基銨之混合物)及苄索氯銨。其他類型之防腐劑包括芳族醇，諸如苯酚、丁醇及苯甲醇；對羥基苯甲酸烷基酯，諸如對羥基苯甲酸甲酯或對羥基苯甲酸丙酯；兒茶酚、間苯二酚、環己醇、3-戊醇及間甲酚。在一個實施例中，本文中之防腐劑為苯甲醇。 如本文所用，術語「治療」係指經設計以改變所治療之個體或細胞在臨床病理學之病程期間的天然病程的臨床介入。所需治療作用包括降低疾病發展速率、改善或減輕疾病病況及緩解或改良預後。舉例而言，若一或多種與癌症相關之症狀減輕或消除，包括(但不限於)降低(或破壞)癌細胞之增殖、減輕由疾病引起之症狀、提高罹患疾病者之生活品質、降低治療疾病所需之其他藥劑的劑量、延緩疾病進展及/或延長個體存活時間，則成功「治療」個體。 如本文所用，「延緩疾病進展」意謂延緩、阻礙、減緩、阻滯、穩定及/或延遲疾病(諸如癌症)之發展。視疾病史及/或所治療之個體而定，此延緩之時間長度可變化。如熟習此項技術者所顯而易知，充分或顯著之延緩實際上可涵蓋預防，其中該個體並不發展該疾病。舉例而言，晚期癌症(諸如轉移之發展)可得到延緩。 「有效量」為至少實現特定病症之可量測改善或預防所需之最小量。本文中之有效量可根據諸如患者之疾病病況、年齡、性別及體重以及抗體引起個體中所需反應之能力的因素而變化。有效量亦為治療有益作用超過治療之任何毒性或有害作用的量。對於預防性用途，有益或所需結果包括諸如消除或減小風險、減輕嚴重程度或延緩疾病發作之結果，該發作包括疾病之生物化學、組織及/或行為症狀，在疾病發展期間呈現之其併發症及中間病理學表型。對於治療用途，有益或所需結果包括諸如降低由疾病引起之一或多種症狀、提高罹患疾病者之生活品質、降低治療疾病所需之其他藥物的劑量、增強另一藥劑之作用(諸如經由靶向)、延緩疾病進展及/或延長存活時間之臨床結果。在癌症或腫瘤之情況下，有效量之藥物可具有以下作用：減少癌細胞數目；減小腫瘤尺寸；抑制(亦即在一定程度上減緩或理想地停止)癌細胞浸潤至周邊器官中；抑制(亦即在一定程度上減緩或理想地停止)腫瘤轉移；在一定程度上抑制腫瘤生長；及/或在一定程度上緩解與病症相關之一或多種症狀。可分一或多次投與來投與有效量。對本發明而言，藥物、化合物或醫藥組合物之有效量為足以直接或間接實現預防性或治療性處理之量。如在臨床背景中所瞭解，藥物、化合物或醫藥組合物之有效量可與或不與另一藥物、化合物或醫藥組合物聯合達成。因此，在投與一或多種治療劑之情形下可考慮「有效劑量」，且若與一或多種其他藥劑聯合，所需結果可達成或已達成，則單一藥劑可視為以有效量給出。 如本文所用，「結合」係指除投與一種治療模式以外還投與另一種治療模式。因此，「結合」係指在對個體執行另一治療模式之前、期間或之後執行一個治療模式。 「病症」為將受益於治療之任何病狀，包括(但不限於)慢性及急性病症或疾病，包括使哺乳動物易患所關注病症之彼等病理病狀。 術語「細胞增殖性病症」及「增殖性病症」係指與某種程度之異常細胞增殖相關之病症。在一個實施例中，細胞增殖性病症為癌症。在一個實施例中，細胞增殖性病症為腫瘤。 如本文所用，「腫瘤」係指不管惡性或良性之所有贅生性細胞生長及增殖，及所有癌前及癌細胞與組織。如本文所提及，術語「癌症」、「癌性」、「細胞增殖性病症」、「增殖性病症」及「腫瘤」不相互排斥。 術語「癌症」及「癌性」係指或描述特徵通常為未經調節細胞生長之哺乳動物生理學病狀。癌症之實例包括(但不限於)癌瘤、淋巴瘤、母細胞瘤、肉瘤及白血病或淋巴惡性疾病。此類癌症之更特定實例包括(但不限於)鱗狀細胞癌(例如上皮鱗狀細胞癌)、肺癌(包括小細胞肺癌、非小細胞肺癌、肺腺癌及肺鱗狀癌)、腹膜癌、肝細胞癌、胃癌(包括胃腸癌及胃腸基質癌)、胰腺癌、神經膠母細胞瘤、子宮頸癌、卵巢癌、肝癌、膀胱癌、泌尿道癌、肝腫瘤、乳癌、結腸癌、直腸癌、結腸直腸癌、子宮內膜癌或子宮癌、唾液腺癌、腎癌、***癌、陰門癌、甲狀腺癌、肝癌、肛門癌、陰莖癌、黑素瘤、淺表散播型黑素瘤、惡性小痣黑素瘤、肢端雀斑樣黑素瘤、結節性黑素瘤、多發性骨髓瘤及B細胞淋巴瘤(包括輕度/濾泡性非霍奇金氏淋巴瘤(non-Hodgkin's lymphoma，NHL)、小淋巴球性(SL)NHL、中度/濾泡性NHL、中度彌漫性NHL、重度免疫母細胞NHL、重度淋巴母細胞NHL、重度小型無裂細胞NHL、巨瘤症NHL、套細胞淋巴瘤、AIDS相關淋巴瘤及瓦爾登斯特倫氏巨球蛋白血症(Waldenstrom's Macroglobulinemia))、慢性淋巴球性白血病(CLL)、急性淋巴母細胞白血病(ALL)、毛細胞白血病、慢性骨髓母細胞白血病及移植後淋巴增生性病症(PTLD)，以及與母斑細胞病相關之異常血管增生、水腫(諸如與腦腫瘤相關之水腫)、梅格斯氏症候群(Meigs' syndrome)、腦癌以及頭頸癌及相關轉移。在某些實施例中，適於用本發明抗體治療之癌症包括乳癌、結腸直腸癌、直腸癌、非小細胞肺癌、神經膠母細胞瘤、非霍奇金氏淋巴瘤(NHL)、腎細胞癌、***癌、肝癌、胰腺癌、軟組織肉瘤、卡堡氏肉瘤(kaposi's sarcoma)、類癌、頭頸癌、卵巢癌、間皮瘤及多發性骨髓瘤。在一些實施例中，癌症係選自：小細胞肺癌、神經膠母細胞瘤、神經母細胞瘤、黑素瘤、乳癌、胃癌、結腸直腸癌(CRC)及肝細胞癌。而在一些實施例中，癌症係選自：非小細胞肺癌、結腸直腸癌、神經膠母細胞瘤及乳癌，包括彼等癌症之轉移性形式。 「化學治療劑」為適用於治療癌症之化合物。化學治療劑之實例包括烷基化劑，諸如噻替派(thiotepa)及環磷醯胺(CYTOXAN® )；磺酸烷基酯，諸如白消安(busulfan)、英丙舒凡(improsulfan)及哌泊舒凡(piposulfan)；氮丙啶，諸如苯唑多巴(benzodopa)、卡波醌(carboquone)、米特多巴(meturedopa)及尤利多巴(uredopa)；乙烯亞胺及甲基三聚氰胺，包括六甲蜜胺(altretamine)、三伸乙基三聚氰胺、三伸乙基磷醯胺、三伸乙基硫代磷醯胺及三羥甲基三聚氰胺；多聚乙醯(尤其布拉他辛(bullatacin)及布拉他辛酮(bullatacinone))；δ-9-四氫***酚(屈***酚(dronabinol)，MARINOL® )；β-拉帕酮(beta-lapachone)；拉帕醇(lapachol)；秋水仙鹼；樺木酸；喜樹鹼(包括合成類似物拓朴替康(topotecan)(HYCAMTIN® )、CPT-11 (伊立替康(irinotecan)，CAMPTOSAR® )、乙醯基喜樹鹼、東莨菪素及9-胺基喜樹鹼)；苔蘚蟲素(bryostatin)；海洋抑素(callystatin)；CC-1065 (包括其阿多來新(adozelesin)、卡折來新(carzelesin)及比折來新(bizelesin)合成類似物)；鬼臼毒素；鬼臼酸；替尼泊甙(teniposide)；念珠藻環肽(尤其念珠藻環肽1及念珠藻環肽8)；海兔毒素(dolastatin)；多卡米辛(duocarmycin)(包括合成類似物KW-2189及CB1-TM1)；艾榴塞洛素(eleutherobin)；水鬼蕉鹼(pancratistatin)；沙考的汀(sarcodictyin)；海綿抑素(spongistatin)；氮芥，諸如苯丁酸氮芥(chlorambucil)、萘氮芥(chlornaphazine)、氯磷醯胺(chlorophosphamide)、雌氮芥(estramustine)、異環磷醯胺(ifosfamide)、甲氮芥(mechlorethamine)、甲氮芥氧化物鹽酸鹽、美法侖(melphalan)、新氮芥(novembichin)、苯芥膽甾醇(phenesterine)、潑尼氮芥(prednimustine)、曲洛磷胺(trofosfamide)、尿嘧啶芥(uracil mustard)；亞硝基脲，諸如卡莫司汀(carmustine)、氯脲菌素(chlorozotocin)、福莫司汀(fotemustine)、洛莫司汀(lomustine)、尼莫司汀(nimustine)及雷莫司汀(ranimnustine)；抗生素，諸如烯二炔抗生素(例如卡奇黴素(calicheamicin)，尤其卡奇黴素γ1I及卡奇黴素ωIl (參見例如Nicolaou等人，Angew. Chem Intl. Ed. Engl. , 33: 183-186 (1994))；CDP323，一種口服α-4整合素抑制劑；達內黴素(dynemicin)，包括達內黴素A；埃斯培拉黴素(esperamicin)；以及新抑癌蛋白發色團及相關色蛋白烯二炔抗生素發色團)；阿克拉黴素(aclacinomysin)；放線菌素(actinomycin)；安麯黴素(authramycin)；偶氮絲胺酸(azaserine)；博來黴素(bleomycin)；放線菌素C(cactinomycin)；卡拉比辛(carabicin)；洋紅黴素(caminomycin)；嗜癌菌素(carzinophilin)；色黴素(chromomycin)；放線菌素d(dactinomycin)；道諾黴素(daunorubicin)；(detorubicin)；6-重氮基-5-側氧基-L-正白胺酸；小紅莓(doxorubicin)(包括ADRIAMYCIN®、(N-嗎啉基)-小紅莓、氰基-(N-嗎啉基)-小紅莓、2-吡咯啉基-小紅莓、小紅莓HCl脂質體注射液(DOXIL®)、脂質小紅莓TLC D-99 (MYOCET®)、聚乙二醇化脂質小紅莓(CAELYX®)及脫氧小紅莓)；表柔比星(epirubicin)；依索比星(esorubicin)；伊達比星(idarubicin)；麻西羅黴素(marcellomycin)；絲裂黴素(mitomycin)，諸如絲裂黴素C；黴酚酸(mycophenolic acid)；諾加黴素(nogalamycin)；橄欖黴素(olivomycin)；培洛黴素(peplomycin)；泊非羅黴素(porfiromycin)；嘌呤黴素(puromycin)；奎那黴素(quelamycin)；羅多比星(rodorubicin)；鏈黑黴素(streptonigrin)；鏈脲佐菌素(streptozocin)；殺結核菌素(tubercidin)；烏苯美司(ubenimex)；淨司他丁(zinostatin)；左柔比星(zorubicin)；抗代謝物，諸如甲胺喋呤(methotrexate)、吉西他濱(gemcitabine)(GEMZAR®)、替加氟(tegafur)(UFTORAL®)、卡培他濱(capecitabine) (XELODA®)、埃坡黴素(epothilone)及5-氟尿嘧啶(5-FU)；考布他汀(combretastatin)；葉酸類似物，諸如迪諾特寧(denopterin)、甲胺喋呤、蝶羅呤(pteropterin)、曲美沙特(trimetrexate)；嘌呤類似物，諸如氟達拉賓(fludarabine)、6-巰基嘌呤、硫米嘌呤(thiamiprine)、硫鳥嘌呤(thioguanine)；嘧啶類似物，諸如安西他濱(ancitabine)、阿紮胞苷(azacitidine)、6-氮尿苷、卡莫氟(carmofur)、阿糖胞苷(cytarabine)、雙脫氧尿苷(dideoxyuridine)、脫氧氟尿苷(doxifluridine)、依諾他濱(enocitabine)、氟尿苷(floxuridine)；雄激素(androgen)，諸如卡魯睾酮(calusterone)、丙酸屈他雄酮(dromostanolone propionate)環硫雄醇(epitiostanol)、美雄烷(mepitiostane)、睾內酯(testolactone)；抗腎上腺藥，諸如胺格魯米特(aminoglutethimide)、米托坦(mitotane)、曲洛司坦(trilostane)；葉酸補充劑，諸如亞葉酸(frolinic acid)；乙醯葡醛酯(aceglatone)；醛磷醯胺醣苷(aldophosphamide glycoside)；胺基乙醯丙酸(aminolevulinic acid)；恩尿嘧啶(eniluracil)；安吖啶(amsacrine)；倍思塔布(bestrabucil)；比生群(bisantrene)；艾達曲克(edatraxate)；地磷醯胺(defofamine)；秋水仙胺(demecolcine)；地吖醌(diaziquone)；艾福米辛(elformithine)；依利醋銨(elliptinium acetate)；埃坡黴素(epothilone)；依託格魯(etoglucid)；硝酸鎵(gallium nitrate)；羥脲；香菇多醣(lentinan)；羅尼達寧(lonidainine)；類美登素(maytansinoid)，諸如美登素(maytansine)及安絲菌素(ansamitocin)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫比達摩(mopidanmol)；硝拉維林(nitraerine)；噴司他丁(pentostatin)；蛋胺氮芥(phenamet)；吡柔比星(pirarubicin)；洛索蒽醌(losoxantrone)；2-乙基醯肼；丙卡巴肼(procarbazine)；PSK®多醣複合物(JHS Natural Products, Eugene, Oreg.)；雷佐生(razoxane)；根瘤菌素(rhizoxin)；西佐喃(sizofuran)；鍺螺胺(spirogermanium)；細交鏈孢菌酮酸(tenuazonic acid)；三亞胺醌(triaziquone)；2,2',2'-三氯三乙胺；單端孢黴烯(尤其T-2毒素、黏液黴素A(verracurin A)、桿孢菌素A(roridin A)及蛇形菌素(anguidine))；烏拉坦(urethan)；長春地辛(vindesine) (ELDISINE®、FILDESIN®)；達卡巴嗪(dacarbazine)；甘露氮芥(mannomustine)；二溴甘露醇(mitobronitol)；二溴衛矛醇(mitolactol)；哌泊溴烷(pipobroman)；加西托星(gacytosine)；***糖苷(arabinoside)(「Ara-C」)；噻替派(thiotepa)；紫杉醇(taxoid)，例如太平洋紫杉醇(paclitaxel)(TAXOL® , Bristol-Myers Squibb Oncology, Princeton, N.J.)、太平洋紫杉醇之白蛋白工程改造奈米粒子調配物(ABRAXANETM )及多西他賽(TAXOTERE® , Rhome-Poulene Rorer, Antony, France)；苯丁酸氮芥；6-硫代鳥嘌呤；巰基嘌呤；甲胺喋呤；鉑試劑，諸如順鉑(cisplatin)、奧沙利鉑(oxaliplatin)(例如ELOXATIN®)及卡鉑(carboplatin)；長春鹼類(vincas)，其防止微管蛋白聚合形成微管，包括長春鹼(vinblastine) (VELBAN®)、長春新鹼(vincristine) (ONCOVIN®)、長春地辛(vindesine) (ELDISINE®、FILDESIN®)及長春瑞賓(vinorelbine)(NAVELBINE®)；依託泊苷(etoposide)(VP-16)；異環磷醯胺；米托蒽醌(mitoxantrone)；甲醯四氫葉酸(leucovorin)；米托蒽醌(novantrone)；依達曲沙(edatrexate)；柔紅黴素(daunomycin)；胺基喋呤(aminopterin)；伊班膦酸鹽(ibandronate)；拓撲異構酶抑制劑(topoisomerase inhibitor) RFS 2000；二氟甲基鳥胺酸(DMFO)；類視黃素，諸如視黃酸，包括貝瑟羅汀(bexarotene) (TARGRETIN®)；雙膦酸鹽，諸如氯屈膦酸鹽(例如BONEFOS®或OSTAC®)、依替膦酸鹽(etidronate) (DIDROCAL®)、NE-58095、唑來膦酸(zoledronic acid)/唑來膦酸鹽(zoledronate) (ZOMETA®)、阿侖膦酸鹽(alendronate) (FOSAMAX®)、帕米膦酸鹽(pamidronate) (AREDIA®)、替魯膦酸鹽(tiludronate)(SKELID®)或利塞膦酸鹽(risedronate)(ACTONEL®)；曲沙他濱(troxacitabine) (1,3-二氧戊環核苷胞嘧啶類似物)；反義寡核苷酸，尤其抑制異常細胞增殖中涉及之信號傳導路徑中的基因表現者，諸如PKC-α、Raf、H-Ras及表皮生長因子受體(EGF-R) (例如埃羅替尼(erlotinib) (TarcevaTM ))；及減少細胞增殖之VEGF-A；疫苗，諸如THERATOPE®疫苗及基因療法疫苗，例如ALLOVECTIN®疫苗、LEUVECTIN®疫苗及VAXID®疫苗；1型拓撲異構酶抑制劑(例如LURTOTECAN®)；rmRH (例如ABARELIX®)；BAY439006 (索拉非尼(sorafenib)；Bayer)；SU-11248 (舒尼替尼(sunitinib)；SUTENT®, Pfizer)；哌立福新(perifosine)；COX-2抑制劑(例如塞內昔布(celecoxib)或依他昔布(etoricoxib))；蛋白酶體抑制劑(例如PS341)；硼替佐米(bortezomib)(VELCADE®)；CCI-779；替吡法尼(tipifarnib) (R11577)；索拉非尼(orafenib)、ABT510；Bcl-2抑制劑，諸如奧利默森鈉(oblimersen sodium) (GENASENSE®)；匹蒽醌(pixantrone)；EGFR抑制劑；酪胺酸激酶抑制劑；絲胺酸-蘇胺酸激酶抑制劑，諸如雷帕黴素(rapamycin) (西羅莫司(sirolimus)，RAPAMUNE®)；法呢基轉移酶抑制劑，諸如洛那法尼(lonafarnib) (SCH 6636, SARASARTM )；及上述任一者的醫藥學上可接受之鹽、酸或衍生物；以及上述兩者或兩者以上之組合，諸如CHOP，環磷醯胺、小紅莓、長春新鹼及潑尼龍之組合療法的縮寫；及FOLFOX，使用奧沙利鉑(ELOXATINTM )與5-FU及甲醯四氫葉酸之組合的治療方案之縮寫，及上述任一者之醫藥學上可接受之鹽、酸或衍生物；以及上述兩者或兩者以上之組合。 如本文所定義，化學治療劑包括用以調控、減少、阻斷或抑制可促進癌症生長之激素之作用的「抗激素劑」或「內分泌治療劑」。其可為激素本身，包括(但不限於)：抗***及選擇性***受體調節劑(SERM)，包括例如他莫昔芬(tamoxifen) (包括NOLVADEX®他莫昔芬)、雷諾昔酚(raloxifene)、曲洛昔芬(droloxifene)、4-羥基他莫昔芬、曲沃昔芬(trioxifene)、雷洛昔芬(keoxifene)、LY117018、奧那司酮(onapristone)及FARESTON.cndot.托瑞米芬(toremifene)；抑制酵素芳香酶之芳香酶抑制劑，其調控腎上腺中之***製造，諸如4(5)-咪唑、胺格魯米特(aminoglutethimide)、MEGASE®乙酸甲地孕酮、AROMASIN®依西美坦(exemestane)、福美斯坦(formestanie)、法屈唑(fadrozole)、RIVISOR®伏羅唑(vorozole)、FEMARA®來曲唑(letrozole)及ARIMIDEX®阿那曲唑(anastrozole)；及抗雄激素，諸如氟他胺(flutamide)、尼魯胺(nilutamide)、比卡魯胺(bicalutamide)、亮丙立德(leuprolide)及戈舍瑞林(goserelin)；以及曲沙他濱(troxacitabine) (1,3-二氧戊環核苷胞嘧啶類似物)；反義寡核苷酸，尤其抑制異常細胞增殖中涉及之信號傳導路徑中的基因表現者，諸如PKC-α、Raf及H-Ras；核糖核酸酶，諸如VEGF表現抑制劑(例如ANGIOZYME®核糖核酸酶)及HER2表現抑制劑；疫苗，諸如基因療法疫苗，例如ALLOVECTIN®疫苗、LEUVECTIN®疫苗及VAXID®疫苗；PROLEUKIN® rIL-2；LURTOTECAN® 1型拓撲異構酶抑制劑；ABARELIX® rmRH；長春瑞賓(Vinorelbine)及埃斯培拉黴素(Esperamicin)(參見美國專利第4,675,187號)，及上述任一者之醫藥學上可接受之鹽、酸或衍生物；以及上述兩者或兩者以上之組合。 「生長抑制劑」當在本文中使用時係指活體外或活體內抑制細胞生長之化合物或組合物。在一個實施例中，生長抑制劑為防止或減少表現抗體所結合之抗原的細胞增殖之生長抑制抗體。在另一實施例中，生長抑制劑可為顯著降低S期中細胞百分比的生長抑制劑。生長抑制劑之實例包括阻斷細胞週期進展(除S期以外之其他位置)之藥劑，諸如誘導G1停滯及M期停滯之藥劑。經典M期阻斷劑包括長春鹼類(長春新鹼及長春鹼)、紫杉烷(taxane)及II型拓撲異構酶抑制劑(諸如小紅莓、表柔比星、柔紅黴素、依託泊苷及博萊黴素)。彼等使G1停滯之藥劑亦深入至S期停滯，例如DNA烷化劑，諸如他莫西芬、潑尼松(prednisone)、氮烯唑胺、甲氮芥、順鉑、甲胺喋呤、5-氟尿啶及ara-C。其他資訊可見於Mendelsohn及Israel編, The Molecular Basis of Cancer, Chapter 1, 題為「Cell cycle regulation, oncogenes, and antineoplastic drugs」, Murakami等人. (W.B. Saunders, Philadelphia, 1995), 例如第13頁。紫杉烷(太平洋紫杉醇及多西他賽)為來源於紫杉樹之抗癌藥物。源自歐洲紫杉之多西他賽(TAXOTERE® , Rhone-Poulenc Rorer)為太平洋紫杉醇(TAXOL® , Bristol-Myers Squibb)之半合成類似物。太平洋紫杉醇及多西他賽促進自微管蛋白二聚體組裝成微管且藉由防止解聚作用而使微管穩定，此將導致抑制細胞中之有絲***。 「放射治療」意謂使用定向γ射線或β射線對細胞誘導足夠破壞，以便限制細胞正常發揮功能之能力或完全破壞細胞。應瞭解，此項技術中已知多種測定治療劑量及持續時間之方式。以單次投與形式給出典型治療且典型劑量在每日10至200單位(戈雷(Gray))之範圍內。 對於治療而言，「個體」係指分類為哺乳動物之任何動物，包括人類、家畜及農畜，及動物園動物、運動動物或寵物動物，諸如犬、馬、貓、牛等。哺乳動物較佳為人類。 本文之術語「抗體」以最廣泛意義使用且尤其涵蓋單株抗體(包括全長單株抗體)、多株抗體、多特異性抗體(例如雙特異性抗體)及抗體片段，只要其展現所要生物活性。 「分離」抗體為已經鑑定且與其自然環境之組分分離且/或自其回收的抗體。其自然環境之污染物組分為干擾抗體研究、診斷性或治療性使用的物質，且可包括酶、激素及其他蛋白性溶質或非蛋白性溶質。在一些實施例中，抗體經純化(1)至如藉由例如洛瑞法(Lowry method)所測定大於95重量%之抗體，且在一些實施例中至大於99重量%；(2)至足以獲得藉由使用例如旋杯式定序儀(spinning cup sequenator)所測定的至少15個N端殘基或內部胺基酸序列之程度；或(3)至藉由使用例如庫馬斯藍(Coomassie blue)或銀染色在還原或非還原條件下進行SDS-PAGE測定為均質之程度。由於抗體之自然環境之至少一種組分將不存在，所以分離之抗體包括重組細胞內之原位抗體。然而，分離之抗體通常將藉由至少一個純化步驟來製備。 「天然抗體」通常為約150,000道爾頓(dalton)之雜四聚體糖蛋白，由兩個相同輕鏈(L)及兩個相同重鏈(H)構成。各輕鏈經一個共價雙硫鍵與重鏈連接，而不同免疫球蛋白同型之重鏈中的雙硫鍵之數目不同。各重鏈及輕鏈亦具有有規律間隔之鏈內雙硫橋。各重鏈在一個末端具有可變域(VH )，其後為大量恆定域。各輕鏈在一個末端具有可變域(VL )且在另一末端具有恆定域；輕鏈之恆定域與重鏈之第一恆定域比對，且輕鏈可變域與重鏈之可變域比對。咸信特定胺基酸殘基在輕鏈與重鏈可變域之間形成界面。 術語「恆定域」係指具有相對於免疫球蛋白其他部分(含有抗原結合位點之可變域)更保守之胺基酸序列的免疫球蛋白分子部分。恆定域含有重鏈之CH 1、CH 2及CH 3結構域(統稱為CH)及輕鏈之CHL (或CL)結構域。 抗體之「可變區」或「可變域」係指抗體之重鏈或輕鏈的胺基端結構域。重鏈之可變域可稱作「VH 」。輕鏈之可變域可稱作「VL 」。此等結構域通常為抗體之最易變部分且含有抗原結合位點。 術語「可變」係指如下事實：可變域之某些部分的序列在各抗體之間廣泛不同且用於各特定抗體對其特定抗原的結合及特異性。然而，可變性在整個抗體可變域中並非均勻分佈。其集中於輕鏈可變域與重鏈可變域中三個稱為高變區(HVR)之區段中。可變域之更高度保守部分稱作構架區(FR)。原生重鏈及輕鏈之可變域各自包含四個主要採用β摺疊構型之FR區，該等FR區由三個HVR連接，該等HVR形成連接β摺疊結構之環，且在一些情況下形成β摺疊結構之一部分。各鏈中之HVR藉由FR區緊密結合在一起以及與其他鏈中之HVR緊密結合在一起，形成抗體之抗原結合位點(參見Kabat等人，Sequences of Proteins of Immunological Interest , 第5版, National Institute of Health, Bethesda, Md. (1991))。恆定域不直接參與抗體與抗原之結合，但展現各種效應功能，諸如使抗體參與抗體依賴性細胞毒性。 來自任何哺乳動物物種的抗體(免疫球蛋白)的「輕鏈」可以基於其恆定域的胺基酸序列分配至兩種稱為κ及λ的明顯不同的類型之一。 如本文所用之術語IgG「同型」或「子類」意謂由恆定區之化學及抗原特徵所限定的免疫球蛋白之任一子類。 抗體(免疫球蛋白)視其重鏈之恆定域之胺基酸序列而定可歸為不同類別。免疫球蛋白有五種主要類別：IgA、IgD、IgE、IgG及IgM，且此等類別中若干類別可進一步分成子類(同型)，例如IgG1 、IgG2 、IgG3 、IgG4 、IgA1 及IgA2 。對應於不同類別免疫球蛋白之重鏈恆定域分別稱作α、δ、ε、γ及μ。不同類別之免疫球蛋白的次單位結構及三維構型為熟知的且一般描述於例如Abbas等人，Cellular and Mol. Immunology , 第4版. (W.B. Saunders, Co., 2000)中。一種抗體可為由該抗體與一或多種其他蛋白質或肽共價或非共價締合形成之較大融合分子的部分。 術語「全長抗體」、「完整抗體」及「全抗體」在本文中可互換使用以指示實質上完整形式之抗體而非如下文所定義之抗體片段。該等術語尤其係指具有含有Fc區之重鏈的抗體。 對本發明而言，「裸抗體」為不與細胞毒性部分或放射性標記結合之抗體。 「抗體片段」包含完整抗體之部分，較佳包含其抗原結合區。在一些實施例中，本文所述之抗體片段為抗原結合片段。抗體片段之實例包括Fab、Fab'、F(ab')2 及Fv片段；雙功能抗體；線性抗體；單鏈抗體分子及由抗體片段形成之多特異性抗體。 抗體之木瓜酶消化產生兩個稱為「Fab」片段之相同抗原結合片段，其各自具有單個抗原結合位點；及一「Fc」片段，該名稱反映其容易結晶之能力。胃蛋白酶處理產生具有兩個抗原組合位點且仍能夠與抗原交聯之F(ab')2 片段。 「Fv」為含有完整抗原結合位點之最小抗體片段。在一個實施例中，雙鏈Fv物質由緊密非共價締合之一個重鏈可變域與一個輕鏈可變域之二聚體組成。在單鏈Fv(scFv)物質中，一個重鏈可變域與一個輕鏈可變域可藉由可撓性肽連接子共價連接，以使得輕鏈與重鏈可以與雙鏈Fv物質類似之「二聚」結構締合。在此構型中，各可變域之三個HVR相互作用以界定VH-VL二聚體表面上之抗原結合位點。總體而言，六個HVR賦予抗體以抗原結合特異性。然而，即使單一可變域(或僅包含三個對抗原具特異性之HVR的一半Fv)亦具有識別且結合抗原之能力，但親和力低於整個結合位點。 Fab片段含有重鏈可變域及輕鏈可變域且亦含有輕鏈之恆定域及重鏈之第一恆定域(CH1)。Fab'片段因在重鏈CH1結構域之羧基端添加少量殘基(包括一或多個來自抗體鉸鏈區之半胱胺酸)而不同於Fab片段。Fab'-SH在本文中指代恆定域之半胱胺酸殘基帶有游離硫醇基之Fab'。F(ab')2 抗體片段最初製造成中間具有鉸鏈區半胱胺酸之Fab'片段對。亦已知抗體片段之其他化學偶合。 「單鏈Fv」或「scFv」抗體片段包含抗體之VH及VL域，其中此等結構域存在於單一多肽鏈中。一般而言，scFv多肽另外包含VH與VL域之間使scFv能夠形成抗原結合所需結構之多肽連接子。scFv之評論參見例如Pluckthün,The Pharmacology of Monoclonal Antibodies , 第113卷, Rosenburg及Moore編, (Springer-Verlag, New York, 1994), 第269頁-第315頁。 術語「雙功能抗體」係指具有兩個抗原結合位點之抗體片段，該等片段包含與同一多肽鏈(VH-VL)中之輕鏈可變域(VL)連接之重鏈可變域(VH)。藉由使用過短而無法使同一鏈上兩個結構域之間配對的連接子，該等結構域被迫與另一鏈之互補結構域配對且產生兩個抗原結合位點。雙功能抗體可為二價或雙特異性的。雙功能抗體更充分描述於例如EP 404,097；WO 1993/01161；Hudson等人，Nat. Med. 9:129-134 (2003)；及Hollinger等人，Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)中。三功能抗體及四功能抗體亦描述於Hudson等人，Nat. Med. 9:129-134 (2003)中。 如本文所用之術語「單株抗體」係指自一群實質上同源抗體獲得之抗體，例如構成該群體之個別抗體除可以微量存在之可能突變(例如天然存在之突變)之外均相同。因此，修飾語「單株」表示抗體當不為離散抗體之混合物時的特性。在某些實施例中，此類單株抗體通常包括包含結合目標之多肽序列之抗體，其中藉由包括自複數個多肽序列選擇單個目標結合多肽序列之方法來獲得目標結合多肽序列。舉例而言，選擇方法可為自複數個純系(諸如融合瘤純系、噬菌體純系或重組DNA純系之庫)中選擇獨特純系。應理解，所選擇之目標結合序列可進一步改變例如以改良對目標之親和力、使目標結合序列人類化、改良其在細胞培養物中之產生、減少其活體內免疫原性、產生多特異性抗體等，且包含經改變之目標結合序列之抗體亦為本發明之單株抗體。與通常包括針對不同決定子(抗原決定基)之不同抗體的多株抗體製劑相對，單株抗體製劑之各單株抗體係針對抗原上之單個決定子。除其特異性外，單株抗體製劑為有利的，此係因為其通常未受到其他免疫球蛋白污染。 修飾語「單株」指示抗體係自實質上均質之抗體群體獲得之特徵，且不應理解為需要藉由任何特定方法產生抗體。舉例而言，待根據本發明使用之單株抗體可藉由多種技術製備，包括例如融合瘤方法(例如Kohler及Milstein,Nature , 256:495-97 (1975)；Hongo等人，Hybridoma , 14 (3): 253-260 (1995)；Harlow等人，Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 第2版. 1988)；Hammerling等人，Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981))；重組DNA方法(參見例如美國專利第4,816,567號)；噬菌體呈現技術(參見例如Clackson等人，Nature , 352: 624-628 (1991)；Marks等人，J. Mol. Biol. 222: 581-597 (1992)；Sidhu等人，J. Mol. Biol. 338(2): 299-310 (2004)；Lee等人，J. Mol. Biol. 340(5): 1073-1093 (2004)；Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004)；及Lee等人，J. Immunol. Methods 284(1-2): 119-132 (2004)；及在動物體內產生具有部分或全部人類免疫球蛋白基因座或編碼人類免疫球蛋白序列之基因的人類或類人類抗體之技術(參見例如WO 1998/24893；WO 1996/34096；WO 1996/33735；WO 1991/10741；Jakobovits等人，Proc. Natl. Acad. Sci. USA 90: 2551 (1993)；Jakobovits等人，Nature 362: 255-258 (1993)；Bruggemann等人，Year in Immunol. 7:33 (1993)；美國專利第5,545,807號；第5,545,806號；第5,569,825號；第5,625,126號；第5,633,425號及第5,661,016號；Marks等人，Bio/Technology 10: 779-783 (1992)；Lonberg等人，Nature 368: 856-859 (1994)；Morrison,Nature 368: 812-813 (1994)；Fishwild等人，Nature Biotechnol. 14: 845-851 (1996)；Neuberger,Nature Biotechnol. 14: 826 (1996)；以及Lonberg及Huszar,Intern. Rev. Immunol. 13: 65-93 (1995)。 本文中之單株抗體尤其包括「嵌合」抗體，其中重鏈及/或輕鏈之一部分與獲自特定物種或屬於特定抗體類別或子類的抗體中之對應序列一致或與其同源，而鏈之剩餘部分與獲自另一物種或屬於另一抗體類別或子類的抗體以及該等抗體之片段中之對應序列一致或與其同源，只要其展示所需生物活性即可(參見例如美國專利第4,816,567號；及Morrison等人，Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984))。嵌合抗體包括PRIMATIZED® 抗體，其中抗體之抗原結合區係獲自藉由例如以所關注抗原使獼猴免疫而產生之抗體。 非人類(例如鼠類)抗體之「人類化」形式為含有最少之源自非人類免疫球蛋白之序列的嵌合抗體。在一個實施例中，人類化抗體為來自接受者之HVR之殘基置換為來自非人類物種(供者抗體)(諸如小鼠、大鼠、兔或非人類靈長類動物)之HVR且具有所要特異性、親和力及/或能力之殘基的人類免疫球蛋白(接受者抗體)。在一些情況下，人類免疫球蛋白之FR殘基置換為相應非人類殘基。此外，人類化抗體可包含接受者抗體或供者抗體中未見之殘基。可進行此等修飾以進一步改進抗體效能。一般而言，人類化抗體將包含實質上全部的至少一個且通常兩個可變域，其中全部或實質上全部高變環對應於非人類免疫球蛋白之高變環，且全部或實質上全部FR為人類免疫球蛋白序列之FR。人類化抗體視情況亦將包含至少一部分免疫球蛋白恆定區(Fc)，通常為人類免疫球蛋白之恆定區。其他細節參見例如Jones等人，Nature 321:522-525 (1986)；Riechmann等人，Nature 332:323-329 (1988)；及Presta，Curr. Op. Struct. Biol. 2:593-596 (1992)。亦參見例如Vaswani及Hamilton,Ann. Allergy, Asthma & Immunol. 1:105-115 (1998)；Harris,Biochem. Soc. Transactions 23:1035-1038 (1995)；Hurle及Gross,Curr. Op. Biotech. 5:428-433 (1994)；以及美國專利第6,982,321號及第7,087,409號。 「人類抗體」為具有對應於由人類產生之抗體的胺基酸序列之胺基酸序列及/或已使用如本文所揭示的用於製備人類抗體之任一技術製備之抗體。人類抗體之此定義尤其排除包含非人類抗原結合殘基之人類化抗體。可使用此項技術中已知之各種技術(包括噬菌體呈現庫)來產生人類抗體。Hoogenboom及Winter,J. Mol. Biol ., 227:381 (1991)；Marks等人，J. Mol. Biol ., 222:581 (1991)。Cole等人，Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, 第77頁 (1985)；Boerner等人，J. Immunol. , 147(1):86-95 (1991)中所述之方法亦可用於製備人類單株抗體。亦參見van Dijk及van de Winkel,Curr. Opin. Pharmacol. , 5: 368-74 (2001)。人類抗體可藉由將抗原投與已經修飾以回應於抗原攻毒產生此類抗體但內源性基因座已不能使用之轉殖基因動物(例如經免疫之異種小鼠(xenomice))來製備(關於XENOMOUSETM 技術參見例如美國專利第6,075,181號及第6,150,584號)。關於經人類B細胞融合瘤技術產生之人類抗體亦參見例如Li等人，Proc. Natl. Acad. Sci. USA , 103:3557-3562 (2006)。 「物種依賴性抗體」為對來自第一哺乳動物物種之抗原的結合親和力比對來自第二哺乳動物物種之該抗原之同系物的結合親和力強的抗體。通常，物種依賴性抗體「特異性結合」人類抗原(例如結合親和力(Kd)值不超過約1×10-7 M，較佳不超過約1×10-8 M且較佳不超過約1×10-9 M)，但對來自第二非人類哺乳動物物種之抗原的同系物之結合親和力比其針對人類抗原之結合親和力弱至少約50倍，或至少約500倍，或至少約1000倍。物種依賴性抗體可為如上文定義之各類型抗體之任一者，但較佳為人類化或人類抗體。 當用於本文中時，術語「高變區」、「HVR」或「HV」係指抗體可變域中序列高變及/或形成結構限定之環的區域。一般而言，抗體包含六個HVR；三個位於VH中(H1、H2、H3)，且三個位於VL中(L1、L2、L3)。在原生抗體中，H3及L3在六個HVR中顯示最具多樣性，且咸信尤其H3在賦予抗體以精細特異性方面起到獨特作用。參見例如Xu等人，Immunity 13:37-45 (2000)；Johnson及Wu,Methods in Molecular Biology 248:1-25 (Lo編, Human Press, Totowa, NJ, 2003)。實際上，僅由重鏈組成之天然存在之駱駝抗體在無輕鏈存在下亦具功能性及穩定性。參見例如Hamers-Casterman等人，Nature 363:446-448 (1993)；Sheriff等人，Nature Struct. Biol. 3:733-736 (1996)。 本文中使用且涵蓋許多HVR敍述。Kabat互補決定區(CDR)係基於序列可變性且最通常使用(Kabat等人，Sequences of Proteins of Immunological Interest ，第5版，Public Health Service, National Institutes of Health, Bethesda, Md. (1991))。Chothia另外指出結構環之位置(Chothia及LeskJ. Mol. Biol. 196:901-917(1987))。AbM HVR表示Kabat HVR與Chothia結構環之間的折衷，且由Oxford Molecular之AbM抗體模型化軟體使用。「接觸」HVR係基於對可用複雜晶體結構之分析。此等HVR中之每一者之殘基註解如下。 環 Kabat AbM Chothia 接觸 L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat編號) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia編號) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101 HVR可包含如下「延長HVR」：VL中之24-36或24-34(L1)、46-56或50-56(L2)及89-97或89-96(L3)，以及VH中之26-35(H1)、50-65或49-65(H2)及93-102、94-102或95-102(H3)。可變域殘基係根據Kabat等人(同上文)關於此等定義中之每一者所述編號。 「構架」或「FR」殘基為除如本文所定義之HVR殘基以外的彼等可變域殘基。 術語「如Kabat中之可變域殘基編號」或「如Kabat中之胺基酸位置編號」及其變化形式係指Kabat等人(同上文)中用於編譯抗體之重鏈可變域或輕鏈可變域的編號系統。使用此編號系統，實際線性胺基酸序列可含有較少或額外胺基酸，相當於簡化或***可變域之FR或HVR。舉例而言，重鏈可變域可包括在H2之殘基52之後的單一胺基酸***(根據Kabat之殘基52a)及在重鏈FR殘基82之後的***殘基(例如根據Kabat之殘基82a、82b及82c等)。可藉由將抗體序列與「標準」Kabat編號序列之同源區相比對，來測定既定抗體中殘基之Kabat編號。 當提及可變域中之殘基(大致輕鏈之殘基1-107及重鏈之殘基1-113)時一般使用Kabat編號系統(例如Kabat等人，Sequences of Immunological Interest . 第5版. Public Health Service, National Institutes of Health, Bethesda, Md. (1991))。當提及免疫球蛋白重鏈恆定區中之殘基時，一般使用「EU編號系統」或「EU索引」(例如Kabat等人(同上文)中所報導之EU索引)。「如Kabat中之EU索引」係指人類IgG1 EU抗體之殘基編號。 表述「線性抗體」係指Zapata等人. (1995Protein Eng , 8(10):1057-1062)中所述之抗體。簡言之，此等抗體包含一對串聯Fd區段(VH-CH1-VH-CH1)，其與互補輕鏈多肽一起形成一對抗原結合區。線性抗體可為雙特異性或單特異性的。 如本文所用，術語「特異性結合」或「具有特異性」係指諸如目標與抗體之間的結合之可量測且可重現之相互作用，其在包括生物分子之分子之異質群體存在下確定目標之存在。舉例而言，特異性結合目標(可為抗原決定基)之抗體為與結合其他目標相比以較大親和力(affinity、avidity)、更容易及/或以更長持續時間結合此目標之抗體。在一個實施例中，如例如藉由放射性免疫分析(RIA)所量測，抗體與無關目標結合之程度為抗體與該目標之結合的約10%以下。在某些實施例中，特異性結合目標之抗體的解離常數(Kd)≤1 μM、≤100 nM、≤10 nM、≤1 nM或≤0.1 nM。在某些實施例中，抗體特異性結合在來自不同物種之蛋白質中為保守的蛋白質上之抗原決定基。在另一實施例中，特異性結合可包括(但並非必需)排他性結合。II. 抗體調配物及製劑 本發明此處係關於穩定水性調配物，其包含抗體，諸如抗PDL1抗體。在一些實施例中，調配物包含抗體(例如單株抗體)、蔗糖、緩衝液及界面活性劑，其中調配物之pH為約5.0至約7.0。在一些實施例中，調配物中之抗體(例如本文所述之抗PDL1抗體)為約40 mg/ml至約125 mg/ml之量。在一些實施例中，緩衝液為組胺酸(例如組胺酸乙酸鹽)或乙酸鈉。在一些實施例中，調配物中之緩衝液為約15 mM至約25 mM之濃度。在一些實施例中，調配物中之蔗糖為約60 mM至約240 mM。在一些實施例中，調配物中之界面活性劑為聚山梨醇酯(例如聚山梨醇酯20)。在一些實施例中，調配物中之聚山梨醇酯為約0.005%(w/v)至約0.06% (w/v)之濃度。在一些實施例中，調配物之pH為約5.0至約6.3。在一些實施例中，本文提供穩定水性醫藥調配物，該調配物包含約40 mg/ml至約125 mg/ml之濃度的抗PDL1單株抗體，約15 mM至約25 mM之濃度的組胺酸乙酸鹽或乙酸鈉，約60 mM至約240 mM之濃度的蔗糖，約0.005%(w/v)至約0.06% (w/v)之濃度的聚山梨醇酯，且pH為約5.0至約6.3。在一些實施例中，調配物包含約125 mg/ml之量的抗PDL1單株抗體，約240 mM之濃度的蔗糖，且pH為約5.5。在一些實施例中，調配物包含約60 mg/ml之量的抗PDL1單株抗體，約120 mM之濃度的蔗糖且pH為約5.8。 在一些實施例中，調配物中之抗體在-20℃下穩定至少約6個月、至少約12個月、至少約18個月、至少兩年、至少三年或至少四年。在一些實施例中，調配物中之抗體在2-8℃下穩定至少約6個月、至少約12個月、至少約18個月、至少兩年或至少三年。在一些實施例中，在儲存後，抗體保留至少約60%、至少約65%、至少約70%、至少約75%、至少約80%、至少約85%、至少約90%或至少約95%其儲存前(亦即醫藥調配物製備時)的生物活性(例如與目標之結合或治療效能)。 在某些實施例中，調配物在約40℃下穩定至少約1天、2天、3天、4天、5天、6天、7天、14天、21天、28天或超過28天。在某些實施例中，調配物在約40℃下穩定至少約1週、2週、3週、4週、5週、6週、7週、8週或超過8週。在某些實施例中，調配物在約25℃下穩定至少1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、13個月、14個月、15個月、16個月、17個月、18個月、19個月、20個月、21個月、22個月、23個月、24個月或超過24個月。在某些實施例中，調配物在約5℃下穩定至少1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、13個月、14個月、15個月、16個月、17個月、 18個月、19個月、20個月、21個月、22個月、23個月、24個月或超過24個月。在某些實施例中，調配物在約-20℃下穩定至少1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、13個月、14個月、15個月、16個月、17個月、18個月、19個月、20個月、21個月、22個月、23個月、24個月、25個月、26個月、27個月、28個月、29個月、30個月、31個月、32個月、33個月、34個月、35個月、36個月、37個月、38個月、39個月、40個月、41個月、42個月、43個月、44個月、45個月、46個月、47個月、48個月或超過48個月。在某些實施例中，調配物在5℃或-20℃下穩定至少1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、12個月、13個月、14個月、15個月、16個月、17個月、18個月、19個月、20個月、21個月、22個月、23個月、24個月、25個月、26個月、27個月、28個月、29個月、30個月、31個月、32個月、33個月、34個月、35個月、36個月、37個月、38個月、39個月、40個月、41個月、42個月、43個月、44個月、45個月、46個月、47個月、48個月或超過48個月。此外，調配物較佳在冷凍(至例如-20℃、-40℃或-70℃)及解凍調配物之後，例如在1、2、3、4或5次冷凍及解凍週期後穩定。A. 抗體 ( 諸如抗 PDL1 抗體 ) 在一些實施例中，調配物中之抗體在重鏈及/或輕鏈序列中包含至少一個色胺酸(例如至少兩個、至少三個或至少四個)。在一些實施例中，胺基酸色胺酸在抗體之CDR區、構架區及/或恆定區中。在一些實施例中，抗體在CDR區中包含兩個或三個色胺酸殘基。在一些實施例中，調配物中之抗體為抗PDL1抗體。PD-L1(漸進式細胞死亡1配位體1)亦稱為PDL1、B7-H1、B7-4、CD274及B7-H，其為跨膜蛋白，且其與PD -1之相互作用抑制T細胞活化及細胞激素產生。在一些實施例中，本文所述之抗PDL1抗體結合至人類PD-L1。可使用本文所述之調配物調配的抗PDL1抗體之實例描述於PCT專利申請案WO 2010/077634 A1及US 8,217,149中，其以引用的方式併入本文中。 在一些實施例中，抗PDL1抗體能夠抑制PD-L1與PD-1之間及/或PD-L1與B7-1之間的結合。在一些實施例中，抗PDL1抗體為單株抗體。在一些實施例中，抗PDL1抗體為選自由以下組成之群的抗體片段：Fab、Fab'-SH、Fv、scFv及(Fab')2 片段。在一些實施例中，抗PDL1抗體為人類化抗體。在一些實施例中，抗PDL1抗體為人類抗體。 描述於WO 2010/077634 A1及US 8,217,149中之抗PDL1抗體可在本文所述之調配物中調配。在一些實施例中，抗PDL1抗體包含含有胺基酸序列SEQ ID NO:30之重鏈可變區，及含有胺基酸序列SEQ ID NO:31之輕鏈可變區。 在一個實施例中，抗PD-L1抗體含有重鏈可變區多肽，其包含HVR-H1、HVR-H2及HVR-H3序列，其中： (a) HVR-H1序列為GFTFSX1 SWIH (SEQ ID NO:11)； (b) HVR-H2序列為AWIX2 PYGGSX3 YYADSVKG (SEQ ID NO:12)； (c) HVR-H3序列為RHWPGGFDY (SEQ ID NO:13)； 另外其中：X1 為D或G；X2 為S或L；X3 為T或S。 在一個特定態樣中，X1 為D；X2 為S且X3 為T。在另一態樣中，多肽進一步包含在根據式：(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的可變區重鏈構架序列。在另一態樣中，構架序列係源自人類共同構架序列。在另一態樣中，構架序列為VH子組III共同構架。在另一態樣中，至少一個構架序列如下： HC-FR1為EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:14) HC-FR2為WVRQAPGKGLEWV (SEQ ID NO:15) HC-FR3為RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4為WGQGTLVTVSA (SEQ ID NO:17)。 在另一態樣中，重鏈多肽進一步與包含HVR-L1、HVR-L2及HVR-L3之可變區輕鏈組合，其中： (a) HVR-L1序列為RASQX4 X5 X6 TX7 X8 A (SEQ ID NO:18)； (b) HVR-L2序列為SASX9 LX10 S (SEQ ID NO:19)； (c) HVR-L3序列為QQX11 X12 X13 X14 PX15 T (SEQ ID NO:20)； 另外其中：X4 為D或V；X5 為V或I；X6 為S或N；X7 為A或F；X8 為V或L；X9 為F或T；X10 為Y或A；X11 為Y、G、F或S；X12 為L、Y、F或W；X13 為Y、N、A、T、G、F或I；X14 為H、V、P、T或I；X15 為A、W、R、P或T。 在另一態樣中，X4 為D；X5 為V；X6 為S；X7 為A；X8 為V；X9 為F；X10 為Y；X11 為Y；X12 為L；X13 為Y；X14 為H；X15 為A。在另一態樣中，輕鏈進一步包含在根據式：(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的可變區輕鏈構架序列。在另一態樣中，構架序列源自人類共同構架序列。在另一態樣中，構架序列為VL κ I共同構架。在另一態樣中，至少一個構架序列如下： LC-FR1為DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2為WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3為GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4為FGQGTKVEIKR (SEQ ID NO:24)。 在另一實施例中，提供經分離之抗PDL1抗體或抗原結合片段，其包含重鏈及輕鏈可變區序列，其中： (a)重鏈包含HVR-H1、HVR-H2及HVR-H3，其中另外： (i) HVR-H1序列為GFTFSX1 SWIH； (SEQ ID NO:11) (ii) HVR-H2序列為AWIX2 PYGGSX3 YYADSVKG (SEQ ID NO:12) (iii) HVR-H3序列為RHWPGGFDY，及 (SEQ ID NO:13) (b) 輕鏈包含HVR-L1、HVR-L2及HVR-L3，其中另外： (i) HVR-L1序列為RASQX4 X5 X6 TX7 X8 A (SEQ ID NO:18) (ii) HVR-L2序列為SASX9 LX10 S；及 (SEQ ID NO:19) (iii) HVR-L3序列為QQX11 X12 X13 X14 PX15 T； (SEQ ID NO:20) 其中另外：X1 為D或G；X2 為S或L；X3 為T或S；X4 為D或V；X5 為V或I；X6 為S或N；X7 為A或F；X8 為V或L；X9 為F或T；X10 為Y或A；X11 為Y、G、F或S；X12 為L、Y、F或W；X13 為Y、N、A、T、G、F或I；X14 為H、V、P、T或I；X15 為A、W、R、P或T。 在一特定態樣中，X1 為D；X2 為S且X3 為T。在另一態樣中，X4 為D；X5 為V；X6 為S；X7 為A；X8 為V；X9 為F；X10 為Y；X11 為Y；X12 為L；X13 為Y；X14 為H；X15 為A。在另一態樣中，X1 為D；X2 為S且X3 為T；X4 為D；X5 為V；X6 為S；X7 為A；X8 為V；X9 為F；X10 為Y；X11 為Y；X12 為L；X13 為Y；X14 為H且X15 為A。 在另一態樣中，重鏈可變區包含一或多個在如(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的構架序列，且輕鏈可變區包含一或多個在如(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的構架序列。在另一態樣中，構架序列源自人類共同構架序列。在另一態樣中，重鏈構架序列源自Kabat子組I、II或III序列。在另一態樣中，重鏈構架序列為VH子組III共同構架。在另一態樣中，一或多個重鏈構架序列如下： HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4 WGQGTLVTVSA (SEQ ID NO:17)。 在另一態樣中，輕鏈構架序列源自Kabat κ I、II、II或IV子組序列。在另一態樣中，輕鏈構架序列為VL κ I共同構架。在另一態樣中，一或多個輕鏈構架序列如下： LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 FGQGTKVEIKR (SEQ ID NO:24)。 在另一特定態樣中，抗體進一步包含人類或鼠類恆定區。在另一態樣中，人類恆定區係選自由以下組成之群：IgG1、IgG2、IgG2、IgG3、IgG4。在另一特定態樣中，人類恆定區為IgG1。在另一態樣中，鼠類恆定區係選自由以下組成之群：IgG1、IgG2A、IgG2B、IgG3。在另一態樣中，鼠類恆定區為IgG2A。在另一特定態樣中，抗體具有減小的或最小效應功能。在另一特定態樣中，最小效應功能由「無效應Fc突變」或非糖基化產生。在另一實施例中，效應子較少Fc突變為恆定區中之N297A或D265A/N297A取代。 在另一實施例中，提供抗PDL1抗體，其包含重鏈及輕鏈可變區序列，其中： (a) 重鏈進一步包含HVR-H1、HVR-H2及HVR-H3序列，其分別與GFTFSDSWIH (SEQ ID NO:25)、AWISPYGGSTYYADSVKG (SEQ ID NO:26)及RHWPGGFDY (SEQ ID NO:13)具有至少85%序列一致性，或 (b) 輕鏈進一步包含HVR-L1、HVR-L2及HVR-L3序列，其分別與RASQDVSTAVA (SEQ ID NO:27)、SASFLYS (SEQ ID NO:28)及QQYLYHPAT (SEQ ID NO:29)具有至少85%序列一致性。 在一特定態樣中，序列一致性為86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%。在另一態樣中，重鏈可變區包含一或多個在如(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的構架序列，且輕鏈可變區包含一或多個在如(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的構架序列。在另一態樣中，構架序列源自人類共同構架序列。在另一態樣中，重鏈構架序列源自Kabat子組I、II或III序列。在另一態樣中，重鏈構架序列為VH子組III共同構架。在另一態樣中，一或多個重鏈構架序列如下： HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4 WGQGTLVTVSA (SEQ ID NO:17)。 在另一態樣中，輕鏈構架序列源自Kabat κ I、II、II或IV子組序列。在另一態樣中，輕鏈構架序列為VL κ I共同構架。在另一態樣中，一或多個輕鏈構架序列如下： LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 FGQGTKVEIKR (SEQ ID NO:24)。 在另一特定態樣中，抗體進一步包含人類或鼠類恆定區。在另一態樣中，人類恆定區係選自由以下組成之群：IgG1、IgG2、IgG2、IgG3、IgG4。在另一特定態樣中，人類恆定區為IgG1。在另一態樣中，鼠類恆定區係選自由以下組成之群：IgG1、IgG2A、IgG2B、IgG3。在另一態樣中，鼠類恆定區為IgG2A。在另一特定態樣中，抗體具有減小的或最小效應功能。在另一特定態樣中，最小效應功能由「無效應Fc突變」或非糖基化產生。在另一實施例中，效應子較少Fc突變為恆定區中之N297A或D265A/N297A取代。 在另一實施例中，提供經分離之抗PDL1抗體，其包含重鏈及輕鏈可變區序列，其中： (a) 重鏈序列具有與重鏈序列：EVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA(SEQ ID NO:30)至少85%序列一致性，或 (b) 輕鏈序列與輕鏈序列：DIQMTQSPSSLSASVGDRVTITCRA SQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:31)具有至少85%序列一致性。 在一特定態樣中，序列一致性為86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%。在另一態樣中，重鏈可變區包含一或多個在如(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的構架序列，且輕鏈可變區包含一或多個在如(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的構架序列。在另一態樣中，構架序列源自人類共同構架序列。在另一態樣中，重鏈構架序列源自Kabat子組I、II或III序列。在另一態樣中，重鏈構架序列為VH子組III共同構架。在另一態樣中，一或多個重鏈構架序列如下： HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4 WGQGTLVTVSA (SEQ ID NO:17)。 在另一態樣中，輕鏈構架序列源自Kabat κ I、II、II或IV子組序列。在另一態樣中，輕鏈構架序列為VL κ I共同構架。在另一態樣中，一或多個輕鏈構架序列如下： LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 FGQGTKVEIKR (SEQ ID NO:24)。 在另一特定態樣中，抗體進一步包含人類或鼠類恆定區。在另一態樣中，人類恆定區係選自由以下組成之群：IgG1、IgG2、IgG2、IgG3、IgG4。在另一特定態樣中，人類恆定區為IgG1。在另一態樣中，鼠類恆定區係選自由以下組成之群：IgG1、IgG2A、IgG2B、IgG3。在另一態樣中，鼠類恆定區為IgG2A。在另一特定態樣中，抗體具有減小的或最小效應功能。在另一特定態樣中，最小效應功能由在原核細胞中產生而產生。在另一特定態樣中，最小效應功能由「無效應Fc突變」或非糖基化產生。在另一實施例中，效應子較少Fc突變為恆定區中之N297A或D265A/N297A取代。 在另一實施例中，提供經分離之抗PDL1抗體，其包含重鏈及輕鏈可變區序列，其中： (a) 重鏈序列具有與重鏈序列：EVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32)至少85%序列一致性，或 (b) 輕鏈序列與輕鏈序列：DIQMTQSPSSLSASVGDRVTITCRASQD VSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:31)具有至少85%序列一致性。 在一特定態樣中，序列一致性為86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%。在另一態樣中，重鏈可變區包含一或多個在如(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的構架序列，且輕鏈可變區包含一或多個在如(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的構架序列。在另一態樣中，構架序列源自人類共同構架序列。在另一態樣中，重鏈構架序列源自Kabat子組I、II或III序列。在另一態樣中，重鏈構架序列為VH子組III共同構架。在另一態樣中，一或多個重鏈構架序列如下： HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4 WGQGTLVTVSS (SEQ ID NO:33)。 在另一態樣中，輕鏈構架序列源自Kabat κ I、II、II或IV子組序列。在另一態樣中，輕鏈構架序列為VL κ I共同構架。在另一態樣中，一或多個輕鏈構架序列如下： LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 FGQGTKVEIKR (SEQ ID NO:24)。 在另一特定態樣中，抗體進一步包含人類或鼠類恆定區。在另一態樣中，人類恆定區係選自由以下組成之群：IgG1、IgG2、IgG2、IgG3、IgG4。在另一特定態樣中，人類恆定區為IgG1。在另一態樣中，鼠類恆定區係選自由以下組成之群：IgG1、IgG2A、IgG2B、IgG3。在另一態樣中，鼠類恆定區為IgG2A。在另一特定態樣中，抗體具有減小的或最小效應功能。在另一特定態樣中，最小效應功能由在原核細胞中產生而產生。在另一特定態樣中，最小效應功能由「無效應Fc突變」或非糖基化產生。在另一實施例中，效應子較少Fc突變為恆定區中之N297A或D265A/N297A取代。 在另一態樣中，重鏈可變區包含一或多個在如(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的構架序列，且輕鏈可變區包含一或多個在如(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的構架序列。在另一態樣中，構架序列源自人類共同構架序列。在另一態樣中，重鏈構架序列源自Kabat子組I、II或III序列。在另一態樣中，重鏈構架序列為VH子組III共同構架。在另一態樣中，一或多個重鏈構架序列如下： HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:34) HC-FR2 WVRQAPGKGLEWVA (SEQ ID NO:35) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4 WGQGTLVTVSS (SEQ ID NO:33)。 在另一態樣中，輕鏈構架序列源自Kabat κ I、II、II或IV子組序列。在另一態樣中，輕鏈構架序列為VL κ I共同構架。在另一態樣中，一或多個輕鏈構架序列如下： LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 FGQGTKVEIK (SEQ ID NO:36)。 在另一特定態樣中，抗體進一步包含人類或鼠類恆定區。在另一態樣中，人類恆定區係選自由以下組成之群：IgG1、IgG2、IgG2、IgG3、IgG4。在另一特定態樣中，人類恆定區為IgG1。在另一態樣中，鼠類恆定區係選自由以下組成之群：IgG1、IgG2A、IgG2B、IgG3。在另一態樣中，鼠類恆定區為IgG2A。在另一特定態樣中，抗體具有減小的或最小效應功能。在另一特定態樣中，最小效應功能由「無效應Fc突變」或非糖基化產生。在另一實施例中，效應子較少Fc突變為恆定區中之N297A或D265A/N297A取代。 在另一實施例中，提供抗PDL1抗體，其包含重鏈及輕鏈可變區序列，其中： (c) 重鏈進一步包含HVR-H1、HVR-H2及HVR-H3序列，其分別與GFTFSDSWIH (SEQ ID NO:4)、AWISPYGGSTYYADSVKG (SEQ ID NO:5)及RHWPGGFDY (SEQ ID NO:6)具有至少85%序列一致性，或 (d) 輕鏈進一步包含HVR-L1、HVR-L2及HVR-L3序列，其分別與RASQDVSTAVA (SEQ ID NO:1)、SASFLYS (SEQ ID NO:2)及QQYLYHPAT (SEQ ID NO:3)具有至少85%序列一致性。 在一特定態樣中，序列一致性為86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%。在另一態樣中，重鏈可變區包含一或多個在如(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4)之HVR之間並列的構架序列，且輕鏈可變區包含一或多個在如(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4)之HVR之間並列的構架序列。在另一態樣中，構架序列係源自人類共同構架序列。在另一態樣中，重鏈構架序列源自Kabat子組I、II或III序列。在另一態樣中，重鏈構架序列為VH子組III共同構架。在另一態樣中，一或多個重鏈構架序列如下： HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:34) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:35) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:16) HC-FR4 WGQGTLVTVSSASTK (SEQ ID NO:33)。 在另一態樣中，輕鏈構架序列源自Kabat κ I、II、II或IV子組序列。在另一態樣中，輕鏈構架序列為VL κ I共同構架。在另一態樣中，一或多個輕鏈構架序列如下： LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 FGQGTKVEIKR (SEQ ID NO:24)。 在另一特定態樣中，抗體進一步包含人類或鼠類恆定區。在另一態樣中，人類恆定區係選自由以下組成之群：IgG1、IgG2、IgG2、IgG3、IgG4。在另一特定態樣中，人類恆定區為IgG1。在另一態樣中，鼠類恆定區係選自由以下組成之群：IgG1、IgG2A、IgG2B、IgG3。在另一態樣中，鼠類恆定區為IgG2A。在另一特定態樣中，抗體具有減小的或最小效應功能。在另一特定態樣中，最小效應功能由「無效應Fc突變」或非糖基化產生。在另一實施例中，效應子較少Fc突變為恆定區中之N297A或D265A/N297A取代。 在另一實施例中，提供經分離之抗PDL1抗體，其包含重鏈及輕鏈可變區序列，其中： (a) 重鏈序列具有與重鏈序列：EVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:8)至少85%序列一致性，或 (b) 輕鏈序列與輕鏈序列：DIQMTQSPSSLSASVGDRVTITCRASQ DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:7)具有至少85%序列一致性。 在一些實施例中，提供包含重鏈及輕鏈可變區序列之經分離抗PDL1抗體，其中輕鏈可變區序列與胺基酸序列SEQ ID NO:7具有至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些實施例中，提供包含重鏈及輕鏈可變區序列之經分離抗PDL1抗體，其中重鏈可變區序列具有與SEQ ID NO:8至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些實施例中，提供包含重鏈及輕鏈可變區序列之經分離抗PDL1抗體，其中輕鏈可變區序列具有與胺基酸序列SEQ ID NO:7至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性且重鏈可變區序列具有與胺基酸序列SEQ ID NO:8至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。 在另一實施例中，提供包含重鏈及輕鏈序列之經分離抗PDL1抗體，其中： (a) 重鏈序列具有與重鏈序列：EVQLVESGGGLVQPGGSLRL SCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:10)至少85%序列一致性，或 (b) 輕鏈序列具有與輕鏈序列：DIQMTQSPSSLSASVGDRVTI TCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:9)至少85%序列一致性。 在一些實施例中，提供包含重鏈及輕鏈序列之經分離之抗PDL1抗體，其中輕鏈序列與胺基酸序列SEQ ID NO:9具有至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些實施例中，提供包含重鏈及輕鏈序列之經分離抗PDL1抗體，其中重鏈序列具有與SEQ ID NO:10至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些實施例中，提供包含重鏈及輕鏈序列之經分離抗PDL1抗體，其中輕鏈序列具有與胺基酸序列SEQ ID NO:9至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性且重鏈序列具有與胺基酸序列SEQ ID NO:10至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。 在一些實施例中，經分離抗PDL1抗體為經氧化單株抗體。在一些實施例中，調配物中之經氧化單株抗體包含含有胺基酸序列SEQ ID NO:9之輕鏈及含有胺基酸序列SEQ ID NO:10之重鏈。在一些實施例中，調配物中之經氧化單株抗體包含含有胺基酸序列SEQ ID NO:10之重鏈，其中W33、W50或W101中之一或多者經氧化。在一些實施例中，調配物中之經氧化單株抗體包含含有胺基酸序列SEQ ID NO:10之重鏈，其中M253及M429中之一或多者經氧化。在一些實施例中，經氧化單株抗體保留至少約60%、至少約65%、至少約70%、至少約75%、至少約80%、至少約85%、至少約90%或至少約95%其儲存前(亦即醫藥調配物製備時)的生物活性(例如與目標之結合或治療效能)。 在一些實施例中，經分離抗PDL1抗體為糖基化單株抗體。在一些實施例中，調配物中之糖基化單株抗體包含含有胺基酸序列SEQ ID NO:9之輕鏈及含有胺基酸序列SEQ ID NO:10之重鏈。在一些實施例中，調配物中之糖基化單株抗體包含含有胺基酸序列SEQ ID NO:10之重鏈，其中一或多個離胺酸經糖基化。在一些實施例中，調配物中之糖基化單株抗體包含含有胺基酸序列SEQ ID NO:10之重鏈，其中K65經糖基化。 在一些實施例中，經分離抗PDL1抗體經去糖基化。 在本文之任何實施例中，經分離抗PDL1抗體可結合於人類PD-L1，例如UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1中所示之人類PD-L1，或其變異體。 在另一實施例中，提供編碼本文所述之任何抗體的經分離核酸。在一些實施例中，核酸進一步包含適用於表現上述抗PDL1抗體中之任一者之核酸的載體。在另一特定態樣中，載體在適於表現核酸之宿主細胞中。在另一特定態樣中，宿主細胞為真核細胞或原核細胞。在另一特定態樣中，真核細胞為哺乳動物細胞，諸如中國倉鼠卵巢細胞(Chinese Hamster Ovary，CHO)。 抗體或其抗原結合片段可使用此項技術中已知之方法(例如藉由包含培養含有核酸之宿主細胞的方法，該核酸編碼上述抗PDL1抗體或抗原結合片段中之任一者)在適於產生此類抗體或片段及回收抗體或片段之條件下以適於表現之形式製備。B. 抗體製備 使用此項技術中可用於產生抗體之技術製備調配物中之抗體，其例示性方法在以下部分中更詳細描述。 抗體係針對所關注抗原(亦即PD-L1，諸如人類PD-L1)。抗原較佳為生物學上重要之多肽，且將抗體投與罹患病症之哺乳動物可在彼哺乳動物中產生治療益處。 (i)抗原製備 視情況結合於其他分子之可溶性抗原或其片段可用作產生抗體之免疫原。對於跨膜分子(諸如受體)，其片段(例如受體之細胞外結構域)可用作免疫原。或者，表現跨膜分子之細胞可用作免疫原。此類細胞可源自天然來源(例如癌細胞株)或可為已藉由重組技術轉形以表現跨膜分子之細胞。適用於製備抗體之其他抗原及其形式對於熟習此項技術者將顯而易知。 (ii)某些基於抗體之方法 多株抗體較佳藉由多次皮下(sc)或腹膜內(ip)注射相關抗原及佐劑而在動物體內產生。其可用於將相關抗原結合至對於待免疫物種為致免疫之蛋白質，例如匙孔螺血氰蛋白、血清白蛋白、牛甲狀腺球蛋白或大豆胰蛋白酶抑制劑，其係使用雙功能或衍生化藥劑進行，例如馬來醯亞胺苯甲醯硫代琥珀醯亞胺酯(藉由半胱胺酸殘基結合)、N-羥基琥珀醯亞胺(藉由離胺酸殘基)、戊二醛、琥珀酸酐、SOCl2 或R1 N=C=NR，其中R及R1 為不同烷基。 藉由將例如100 μg或5 μg蛋白質或結合物(分別針對兔或小鼠)與3體積弗氏完全佐劑(Freund's complete adjuvant)組合且在多個部位皮內注射該溶液使動物對抗原、免疫原性結合物或衍生物免疫。一個月後，在多個部位以1/5至1/10原始量之弗氏完全佐劑中之肽或結合物皮下注射來對動物進行加打。7至14天後對動物進行放血且分析血清之抗體力價。對動物進行加打直至力價平穩。較佳地，動物經相同抗原(但該抗原與不同蛋白質結合及/或經不同交聯劑結合)之結合物加打。結合物亦可在重組細胞培養物中作為蛋白質融合物產生。同樣，諸如明礬之聚集劑適用於提高免疫反應。 本發明之單株抗體可使用融合瘤方法初次由Kohler等人，Nature , 256:495 (1975)描述，且進一步描述於例如關於人類-人類融合瘤之Hongo等人，Hybridoma , 14 (3): 253-260 (1995)；Harlow等人，Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 第2版. 1988)；Hammerling等人，Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)；及Ni,Xiandai Mianyixue , 26(4):265-268 (2006)中之融合瘤方法製造。其他方法包括例如美國專利第7,189,826號中關於自融合瘤細胞株產生單株人類天然IgM抗體所描述之彼等方法。人類融合瘤技術(三體雜交瘤(Trioma)技術)亦描述Vollmers及Brandlein,Histology and Histopathology , 20(3):927-937 (2005)以及Vollmers及Brandlein,Methods and Findings in Experimental and Clinical Pharmacology , 27(3):185-91 (2005)中。 多種其他融合瘤技術參見例如US 2006/258841；US 2006/183887 (完全人類抗體)；US 2006/059575；US 2005/287149；US 2005/100546；US 2005/026229；及美國專利第7,078,492號及第7,153,507號。使用融合瘤方法製造單株抗體之例示性方案如下文所述。在一個實施例中，對小鼠或其他適當宿主動物(諸如倉鼠)進行免疫以引起產生或能夠產生特異性結合用於免疫之蛋白質之抗體的淋巴細胞。藉由多次皮下(sc)或腹膜內(ip)注射本發明之多肽或其片段及佐劑(諸如單磷醯基脂質A (MPL)/海藻糖二氰基梅菌酸酯(TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.))而在動物體內產生抗體。可使用此項技術中熟知之方法(諸如重組方法)製備本發明之多肽(例如抗原)或其片段，其中一些方法在本文中進一步描述。分析來自經免疫動物之血清中的抗-抗原抗體，且視情況投與增強免疫。分離出來自產生抗-抗原抗體之動物的淋巴細胞。或者，可活體外使淋巴細胞免疫。 隨後使用適合融合劑(諸如聚乙二醇)使淋巴細胞與骨髓瘤細胞融合形成融合瘤細胞。參見例如Goding,Monoclonal Antibodies: Principles and Practice , 第59頁-第103頁 (Academic Press, 1986)。可使用有效融合、支持藉由所選抗體產生細胞穩定高含量產生抗體且對諸如HAT培養基之培養基敏感的骨髓瘤細胞。例示性骨髓瘤細胞包括(但不限於)鼠類骨髓瘤細胞株，諸如源自獲自Salk Institute Cell Distribution Center, San Diego, Calif. USA之MOPC-21及MPC-11小鼠腫瘤及源自American Type Culture Collection, Rockville, Md. USA之SP-2或X63-Ag8-653細胞者。亦已針對人類單株抗體之產生描述人類骨髓瘤及小鼠-人類雜交骨髓瘤細胞株(Kozbor,J. Immunol. , 133:3001 (1984)；Brodeur等人，Monoclonal Antibody Production Techniques and Applications, 第51頁-第63頁 (Marcel Dekker, Inc., New York, 1987))。 接種由此製備之融合瘤細胞且使其在適合培養基(例如含有一或多種抑制未融合之親本骨髓瘤細胞之生長或存活的物質的培養基)中生長。舉例而言，若親本骨髓瘤細胞缺乏酵素次黃嘌呤鳥嘌呤磷酸核糖轉移酶(HGPRT或HPRT)，則用於融合瘤之培養基中通常將包括次黃嘌呤、胺基蝶呤及胸苷(HAT培養基)，該等物質會阻止HGPRT缺陷細胞之生長。較佳地，如例如Even等人，Trends in Biotechnology , 24(3), 105-108 (2006)中所描述，使用不含血清之融合瘤細胞培養方法來減少源自動物之血清(諸如胎牛血清)的使用。 作為提高融合瘤細胞培養物之產生率之工具的寡肽描述於Franek,Trends in Monoclonal Antibody Research , 111-122 (2005)中。特定言之，標準培養基富含某些胺基酸(丙胺酸、絲胺酸、天冬醯胺、脯胺酸)或蛋白質水解產物部分，且可藉由由三個至六個胺基酸殘基構成之合成寡肽顯著抑制細胞雕亡。該等肽以毫莫耳濃度或較高濃度存在。 可分析生長融合瘤細胞之培養基中結合至本發明抗體之單株抗體的產生。可藉由免疫沈澱或藉由活體外結合分析(諸如放射免疫分析(RIA)或酶聯免疫吸附分析(ELISA))測定由融合瘤細胞產生之單株抗體的結合特異性。可藉由例如史卡查分析(Scatchard analysis)來測定單株抗體之結合親和力。參見例如Munson等人，Anal. Biochem. , 107:220 (1980)。 在鑑別出產生具有所要特異性、親和力及/或活性之抗體的融合瘤細胞之後，可藉由限制性稀釋程序對純系進行次選殖且藉由標準方法使其生長。參見，例如Goding，同上文。適用於此目的之適合培養基包括例如D-MEM或RPMI-1640培養基。另外，可在活體內在動物中以腹水腫瘤形式培養融合瘤細胞。由次純系分泌之單株抗體係藉由習知免疫球蛋白純化程序(諸如蛋白質A-瓊脂糖凝膠、羥磷灰石層析、凝膠電泳、透析或親和力層析)適當地與培養基、腹水流體或血清分離。一種自融合瘤細胞分離蛋白質之程序描述於US 2005/176122及美國專利第6,919,436號中。該方法包括在結合過程中使用最低限度之鹽(諸如易溶鹽)及亦較佳地在溶離過程中使用少量有機溶劑。 (iii)某些文庫篩選法 可藉由使用組合文庫篩選具有所需活性之抗體來製備本發明之抗體。舉例而言，此項技術中已知多種方法可用於產生噬菌體呈現文庫及篩選此類文庫中具有所需結合特徵之抗體。此類方法一般描述於Hoogenboom等人之Methods in Molecular Biology 178:1-37(O'Brien等人編，Human Press, Totowa, NJ, 2001)中。舉例而言，如Lee等人，J. Mol. Biol. (2004), 340(5):1073-93中所述，一種產生所關注抗體之方法係藉由使用噬菌體抗體文庫進行。 大體上，藉由篩選含有呈現融合至噬菌體鞘蛋白之抗體可變區(Fv)之各種片段之噬菌體的噬菌體文庫來選擇合成抗體純系。藉由對所需抗原進行親和力層析來篩檢此類噬菌體文庫。使表現能夠與所需抗原結合之Fv片段之純系吸附至抗原且因此使其與文庫中非結合純系分離。接著將結合純系自抗原溶離，且可藉由額外之抗原吸附/溶離循環進一步增濃。可藉由設計適合抗原篩選程序以選擇所關注噬菌體純系，接著使用來自所關注噬菌體純系之Fv序列及Kabat等人， Sequences of Proteins of Immunological Interest , 第5版, NIH Publication 91-3242, Bethesda MD (1991), 第1-3卷中所描述之適合恆定區(Fc)序列來構築全長抗體純系，從而獲得本發明之任何抗體。 在某些實施例中，抗體之抗原結合域係由兩個具有約110個胺基酸之可變(V)區形成，各自分別來自輕鏈(VL)及重鏈(VH)，兩者皆呈現三個高變環(HVR)或互補決定區(CDR)。可變域可如單鏈Fv(scFv)片段(其中VH與VL經由柔性短肽以共價鍵聯結)或如Fab片段(其中VH與VL各與恆定域融合且以非共價鍵相互作用)，功能性地呈現於噬菌體上，如Winter等人，Ann. Rev. Immunol. , 12: 433-455(1994)中所述。如本文中所用，編碼scFv之噬菌體純系及編碼Fab之噬菌體純系統稱為「Fv噬菌體純系」或「Fv純系」。 VH與VL基因庫可藉由聚合酶鏈反應(PCR)分別選殖，且在噬菌體文庫中隨機重組，接著可搜尋抗原結合純系，如Winter等人，Ann. Rev. Immunol. , 12: 433-455(1994)中所述。來自經免疫來源之文庫無需構築融合瘤即可提供免疫原之高親和力抗體。或者，天然文庫經選殖可向廣範圍之非自體抗原以及自體抗原提供單一來源之人類抗體而無需任何免疫作用，如Griffiths等人，EMBO J, 12: 725-734 (1993)所述。最後，天然文庫亦可藉由自幹細胞選殖未經重排之V-基因片段且使用含有隨機序列之PCR引子編碼高變CDR3區及完成活體外重排來合成獲得，如Hoogenboom及Winter,J. Mol. Biol. , 227: 381-388 (1992)所述。 在某些實施例中，使用絲狀噬菌體藉由融合於次要鞘蛋白pIII來呈現抗體片段。該等抗體片段可作為單鏈Fv片段呈現，其中VH域與VL域藉由柔性多肽間隔基連接於同一多肽鏈上，例如如Marks等人，J. Mol. Biol. , 222:581-597(1991)中所述；或作為Fab片段呈現，其中一鏈與pIII融合而另一鏈分泌進入細菌性宿主細胞周質內，在周質內藉由置換野生型外被蛋白中之一部分組裝呈現於噬菌體表面上的Fab-外被蛋白結構，例如如Hoogenboom等人，Nucl. Acids Res., 19 : 4133-4137(1991)中所述。 大體而言，編碼抗體基因片段之核酸可自由人類或動物採集之免疫細胞獲得。若需要偏重於抗-抗原純系之文庫，則用抗原使個體免疫以產生抗體反應，且可回收脾細胞及/或循環B細胞及其他周圍血液淋巴細胞(PBL)用於文庫構築。在一個實施例中，偏重於抗-抗原純系之人類抗體基因片段文庫係藉由在攜有功能性人類免疫球蛋白基因陣列(且缺乏功能性內源性抗體產生系統)之轉殖基因小鼠中產生抗-抗原抗體反應以便抗原免疫使B細胞產生抗抗原之人類抗體來獲得。下文將描述產生人類抗體之轉殖基因小鼠的產生。 可藉由使用適合篩選程序分離表現抗原特異性膜結合抗體之B細胞(例如藉由用抗原親和力層析分離細胞或使細胞吸附至經螢光染料標記之抗原隨後進行流式活化細胞分選(FACS))來獲得抗-抗原反應性細胞群體之額外增濃。 或者，使用來自未經免疫供體之脾細胞及/或B細胞或其他PBL以使可能之抗體圖譜得以更佳呈現，且亦允許使用抗原不具抗原性之任何動物(人類或非人類)物種來構築抗體文庫。對於併入活體外抗體基因構築之文庫，自個體採集幹細胞以提供編碼未經重排之抗體基因區段之核酸。所關注之免疫細胞可自多種動物物種獲得，諸如人類、小鼠、大鼠、兔類、狼、犬科、貓科、豬、牛、馬及鳥類物種等。 自所關注之細胞中回收編碼抗體可變基因區段(包括VH及VL區段)之核酸且使其擴增。在重排VH及VL基因文庫之狀況中，所要DNA可藉由自淋巴細胞中分離基因組DNA或mRNA、再利用與重排VH及VL基因之5'端及3'端匹配之引子、藉由聚合酶鏈反應(PCR)獲得，如Orlandi等人，Proc. Natl. Acad. Sci. (USA), 86:3833-3837 (1989)中所述，從而獲得用於表現之各種V基因文庫。V基因可利用編碼成熟V-域之外顯子之5'端處的後向引子以及定位於J-區段內部的前向引子，自cDNA及基因組DNA擴增，如Orlandi等人 (1989)及Ward等人，Nature , 341:544-546 (1989)中所述。然而，對於自cDNA擴增，亦可使後向引子定位於前導外顯子中，如Jones等人，Biotechnol. , 9:88-89 (1991)中所述，而前向引子亦可定位於恆定區內部，如Sastry等人，Proc. Natl. Acad. Sci. (USA) , 86:5728-5732 (1989)。為使互補性最大化，可將簡併整合入該等引子中，如Orlandi等人(1989)或Sastry等人(1989)中所述。在某些實施例中，文庫多樣性可藉由使用靶向各V-基因家族的PCR引子最大化，以便增加存在於免疫細胞核酸樣本中之所有可利用的VH與VL排列，例如，如Marks等人，J. Mol. Biol. , 222:581-597 (1991)之方法中所述，或如Orum等人，Nucleic Acids Res. , 21:4491-4498 (1993)之方法中所述。對於將經擴增之DNA選殖入表現載體，可在一端處作為標記之PCR引子內引入稀少的限制性位點，如Orlandi等人(1989)中所述；或利用標記引子進行其他PCR擴增，如Clackson等人，Nature , 352: 624-628 (1991)中所述。 合成性重排之V基因文庫可於活體外自V基因區段獲得。人類VH-基因區段中的大部分已經選殖且定序(報導於Tomlinson等人，J. Mol. Biol. , 227:776-798 (1992)中)，且已繪出圖譜(報導於Matsuda等人，Nature Genet. ,3 :88-94 (1993)中)；可使用此等選殖區段(包括H1與H2環之所有主要構形)以及編碼各種序列及長度之H3環的PCR引子產生各種VH基因文庫，如Hoogenboom及Winter,J. Mol. Biol. , 227:381-388 (1992)中所述。亦可獲得所有的序列多樣性集中於單倍長度之長H3環的VH文庫，如Barbas等人，Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992)中所述。人類Vκ及Vλ區段已經選殖及定序(報導於Williams及Winter,Eur. J. Immunol. , 23:1456-1461 (1993)中)且可用於獲得合成性輕鏈文庫。基於一定範圍之VH與VL摺疊及L3與H3長度之合成，V基因文庫將編碼具有大量結構多樣性之抗體。擴增編碼DNA之V-基因之後，生殖系V-基因區段可根據Hoogenboom及Winter,J. Mol. Biol. , 227: 381-388 (1992)之方法活體外重排。 可使用數種方式藉由將VH與VL基因文庫組合在一起來構築抗體片段文庫。各文庫可在不同載體中產生，且載體活體外重組(例如Hogrefe等人，Gene , 128: 119-126 (1993)中所述)或藉由組合感染活體內重組(例如Waterhouse等人，Nucl. Acids Res. , 21: 2265-2266 (1993)中所述之loxP系統)。活體內重組方法利用Fab片段之雙鏈性質來克服大腸桿菌轉形功率所強加之對於文庫大小之限制。各別地選殖原生VH及VL文庫，將一個選殖至噬菌粒中而另一個選殖至噬菌體載體中。接著藉由含有噬菌粒之細菌的噬菌體感染來組合兩個文庫，從而使各細胞含有不同組合且使文庫大小僅受所存在細胞數目(約1012 個純系)限制。兩載體皆含有活體內重組信號，從而使VH與VL基因重組於單一複製子上且共包裝於噬菌體病毒粒子中。此等大型文庫提供大量具有良好親和力(Kd -1 為約10-8 M)之多種抗體。 或者，該等文庫可依序選殖入同一載體，例如如Barbas等人，Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991)中所述；或藉由PCR組裝在一起且接著選殖，例如如Clackson等人，Nature, 352:624-628 (1991)中所述。亦可使用PCR組裝將VH及VL DNA與編碼柔性肽間隔基之DNA連接在一起以形成單鏈Fv(scFv)文庫。在另一技術中，「在細胞PCR總成中」用於在淋巴細胞內藉由PCR組合VH及VL基因，接著如Embleton等人，Nucl. Acids Res. , 20: 3831-3837 (1992)中所述選殖鍵聯基因之文庫。 由原生文庫所產生之抗體(天然的或合成的)可具有中度親和力(約106 至107 M-1 之Kd -1 )，但親和力成熟亦可藉由構築第二文庫及從中再選擇來模擬，如Winter等人(1994)(同上文)中所述。舉例而言，可藉由使用易錯聚合酶(Leung等人，Technique 1: 11-15 (1989)中報導)使用Hawkins等人，J. Mol. Biol., 226: 889-896 (1992)之方法或Gram等人，Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992)之方法在隨機位置活體外引入突變。此外，可藉由例如用攜帶跨越所關注CDR之隨機序列的引子使用PCR在所選個別Fv純系中使一或多個CDR隨機突變且篩選較高親和力純系來進行親和力成熟。WO 9607754(1996年3月14日公開)描述一種誘導免疫球蛋白輕鏈之互補決定區發生突變以形成輕鏈基因文庫的方法。另一有效方法係將藉由噬菌體呈現所選擇的VH域或VL域與獲自未免疫供體之天然發生V域變體文庫重組且以數輪之鏈改組篩選較高親和力，如Marks等人，Biotechnol. , 10:779-783 (1992)中所述。此技術使得可產生親和力為約10-9 M或10-9 M以下之抗體及抗體片段。 可藉由此項技術中已知之各種技術篩選文庫。舉例而言，可使用抗原塗佈吸附板之孔，表現於附著於吸附板上之宿主細胞上或用於細胞分選，或結合於生物素以供塗佈抗生蛋白鏈菌素之珠粒捕捉，或用於任何其他淘選噬菌體呈現文庫之方法中。 在適於使噬菌體顆粒之至少一部分與吸附劑結合之條件下，使噬菌體文庫樣本與經固定之抗原接觸。一般而言，對包括pH、離子強度、溫度及其類似條件之條件進行選擇以模擬生理條件。將結合固相之噬菌體洗滌且接著藉由酸溶離，例如如Barbas等人，Proc. Natl. Acad. Sci USA , 88:7978-7982 (1991)中所述；或藉由鹼溶離，例如如Marks等人，J. Mol. Biol. , 222:581-597 (1991)中所述；或藉由抗原競爭(例如以與Clackson等人，Nature , 352:624-628 (1991)之抗原競爭法類似之程序)。可在單輪選擇中增濃20-1,000倍之噬菌體。此外，可使所增濃之噬菌體在細菌培養物中生長且經歷另一輪選擇。 選擇效率視許多因素而定，包括洗滌期間之解離動力學及單一噬菌體上之多個抗體片段能否同時與抗原接合。具有快速解離動力學(及弱結合親和力)之抗體可藉由使用短時間洗滌、多價噬菌體呈現及抗原於固相中之高塗覆密度予以保留。高密度不僅藉由多價相互作用使噬菌體穩定，且亦有利於使已解離之噬菌體再結合。具有緩慢解離動力學(及良好結合親和力)之抗體選擇可藉由使用長時間洗滌及單價噬菌體呈現(如Bass等人，Proteins , 8: 309-314 (1990)及WO 92/09690中所述)以及低抗原塗佈密度(如Marks等人，Biotechnol., 10: 779-783 (1992)中所述)促進。 有可能在對抗原具有不同親和力、甚至具有略微不同親和力的噬菌體抗體之間進行選擇。然而，所選抗體之隨機突變(例如如在一些親和力成熟技術中所執行)可能產生許多突變體，其中大多數結合至抗原上且少數具有較高親和力。使用限制性抗原可競爭淘汰極少之高親和力噬菌體。為了保留所有較高親和力突變體，可將噬菌體與過量經生物素標記之抗原一起培育，但經生物素標記之抗原的莫耳濃度比抗原之目標莫耳親和力常數低。接著可以經抗生蛋白鏈菌素塗佈之順磁珠理捕捉高親和力結合之噬菌體。此類「平衡捕集」容許抗體根據其結合親和力選擇，其敏感度容許自顯著過量之具有較低親和力之噬菌體中分離僅兩倍高親和力的突變體純系。亦可操控洗滌與固相結合之噬菌體中所用的條件以基於解離動力學進行區分。 可基於活性選擇抗-抗原純系。在某些實施例中，本發明提供結合至天然地表現抗原之活細胞或結合至自由漂浮抗原或連接於其他細胞結構之抗原的抗-抗原抗體。對應於此類抗-抗原抗體的Fv純系可藉由如下步驟選擇：(1)如上文所述自噬菌體文庫中分離抗-抗原純系，且視情況藉由在適合細菌宿主中培育該群體來擴增噬菌體純系之分離群體；(2)針對分別需要阻斷活性及非阻斷活性來選擇抗原及第二蛋白；(3)將抗-抗原噬菌體純系吸附至固定抗原上；(4)使用過量第二蛋白溶離任何識別與第二蛋白之結合決定子重疊或共有之抗原結合決定子的非所要純系；及(5)溶離步驟(4)完成後仍吸附的純系。視情況而言，具有所要阻斷/非阻斷特性之純系可進一步藉由將本文中所述之選擇程序重複一或多次來增濃。 容易分離編碼本發明之源自融合瘤之單株抗體或噬菌體呈現Fv純系之DNA，且使用習知程序(例如藉由使用經設計以自融合瘤或噬菌體DNA模板特異性擴增相關重鏈及輕鏈編碼區之寡核苷酸引子)進行定序。分離後，可將DNA置於表現載體內，接著將表現載體轉染至不會另外產生免疫球蛋白之宿主細胞(諸如大腸桿菌細胞、猿COS細胞、中國倉鼠卵巢(CHO)細胞或骨髓瘤細胞)中以於重組宿主細胞中獲得所需單株抗體之合成。關於在細菌中重組表現編碼抗體之DNA的評論文章包括Skerra等人，Curr. Opinion in Immunol. , 5: 256 (1993)及Pluckthun,Immunol. Revs , 130: 151 (1992)。 可將編碼本發明Fv純系之DNA與已知編碼重鏈及/或輕鏈恆定區之DNA序列(例如適當DNA序列可由Kabat等人(同上文)獲得)組合以形成編碼全長或部分長度重鏈及/或輕鏈之純系。應瞭解為實現此目的可使用任何同型之恆定區，包括IgG、IgM、IgA、IgD及IgE恆定區，且此類恆定區可自任何人類或動物物種獲得。源自一種動物(諸如人類)物種之可變域DNA且隨後與另一動物物種之恆定區DNA融合以形成「雜交」全長重鏈及/或輕鏈之編碼序列的Fv純系包括於如本文所用之「嵌合」及「雜交」抗體之定義中。在某些實施例中，源自人類可變DNA的Fv純系融合於人類恆定區DNA形成全長或部分長度人類重鏈及/或輕鏈的編碼序列。 編碼本發明之獲自雜交瘤之抗-抗原抗體的DNA亦可經修飾，例如用人類重鏈恆定域及輕鏈恆定域編碼序列取代源自雜交瘤純系的同源鼠類序列(例如，用Morrison等人，Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984)之方法)。可藉由使非免疫球蛋白多肽之所有或部分編碼序列共價接合於免疫球蛋白編碼序列來進一步修飾編碼源自融合瘤或Fv純系之抗體或片段的DNA。可以此方式製得具有本發明之Fv純系之結合特異性或源自雜交瘤純系之抗體之結合特異性的「嵌合」或「雜交」抗體。 (iv) 人類化及人類抗體 人類化非人類抗體的多種方法在此項技術中已知。舉例而言，人類化抗體具有一或多個從非人類來源引入到其中的胺基酸殘基。此等非人類胺基酸殘基通常稱為「輸入」殘基，其通常取自「輸入」可變域。人類化可基本上遵照Winter及合作者之方法(Jones等人，Nature , 321:522-525 (1986)；Riechmann等人，Nature , 332:323-327 (1988)；Verhoeyen等人，Science , 239:1534-1536 (1988))，藉由用嚙齒動物CDR或CDR序列取代人類抗體之相應序列來執行。因此，此類「人類化」抗體為嵌合抗體(美國專利第4,816,567號)，其中實質上少於完整人類可變域之可變域經來自非人類物種之相應序列取代。實際上，人類化抗體通常為一些CDR殘基及可能的一些FR殘基經來自嚙齒動物抗體之類似位點之殘基取代的人類抗體。 欲用於製造人類化抗體之人類可變域之備選者(輕鏈與重鏈)對降低抗原性極其重要。根據所謂「最佳擬合」法，相對於整個已知人類可變域序列文庫篩選嚙齒動物抗體之可變域序列。接著，接受最接近嚙齒動物序列之人類序列作為人類化抗體之人類構架(FR)(參見Sims等人，J. Immunol ., 151:2296 (1993)；Chothia等人，J. Mol. Biol ., 196:901 (1987))。另一方法使用自具有特定輕鏈或重鏈子組之所有人類抗體之共同序列獲得的特定構架。相同構架可用於若干不同人類化抗體(Carter等人，Proc. Natl. Acad Sci. USA , 89:4285 (1992)；Presta等人，J. Immunol. , 151:2623 (1993))。 更重要的是，使抗體人類化且保留對抗原之高親和力及其他有利生物特性。為實現此目標，根據該方法之一個實施例，藉由使用親本序列及人類化序列之三維模型分析親本序列及各種概念上之人類化產物的方法來製備人類化抗體。三維免疫球蛋白模型普遍可得且為熟習此項技術者熟悉。可獲得說明及呈現所選候選免疫球蛋白序列之可能三維構形結構的電腦程式。對此等呈現之檢查允許分析殘基在候選免疫球蛋白序列起作用過程中可能的作用，亦即分析影響候選免疫球蛋白結合其抗原之能力的殘基。以此方式，可自接受者及輸入序列選擇FR殘基且與接受者及輸入序列組合以便獲得所需抗體特徵，諸如對目標抗原之親和力增加。一般而言，高變區殘基直接且多數實質上涉及影響抗原結合。 可藉由將選自源自人類之噬菌體呈現庫之Fv純系可變域序列與如上文所述之已知人類恆定域序列組合來構築本發明之人類抗體。或者，可由融合瘤方法製備本發明之人類單株抗體。用於製造人類單株抗體之人類骨髓瘤及小鼠-人類雜交骨髓瘤細胞株已例如由KozborJ. Immunol. , 133: 3001 (1984)；Brodeur等人，Monoclonal Antibody Production Techniques and Applications , 第51頁-第63頁 (Marcel Dekker, Inc., New York, 1987)；及Boerner等人，J. Immunol. , 147: 86 (1991)描述。 有可能產生在不存在內源性免疫球蛋白產生之情況下一旦免疫即能產生人類抗體全文庫的轉殖基因動物(例如小鼠)。舉例而言，已描述嵌合及生殖系突變小鼠中抗體重鏈連接區(JH )基因的純合子缺失導致對於內源抗體產生之完全抑制。將人類生殖系免疫球蛋白基因陣列轉移至此類生殖系突變體小鼠體內將會於抗原攻擊後引起人類抗體之產生。參見例如Jakobovits等人，Proc. Natl. Acad. Sci. USA , 90:2551 (1993)；Jakobovits等人，Nature , 362:255-258 (1993)；Bruggermann等人，Year in Immuno. , 7:33 (1993)；及Duchosal等人，Nature 355:258 (1992)。 亦可使用基因改組自非人類(例如嚙齒動物)抗體獲得人類抗體，其中人類抗體具有與初始非人類抗體類似之親和力及特異性。根據此方法，亦稱為「抗原決定基印記」，由如本文所述之噬菌體呈現技術獲得之非人類抗體片段之重鏈或輕鏈可變區經人類V域基因文庫置換，形成非人類鏈/人類鏈scFv或Fab嵌合體之群體。使用抗原進行選擇可導致非人類鏈/人類鏈嵌合scFv或Fab之分離，其中人類鏈恢復移除初級噬菌體呈現純系中之相應非人類鏈時損壞的抗原結合位點，亦即抗原決定基決定(印模)對於人類鏈搭配物之選擇。當重複該方法以置換剩餘非人類鏈時，獲得人類抗體(參見1993年4月1日公開之PCT WO 93/06213號)。與藉由CDR移植非人類抗體之傳統人類化不同，此技術提供不具有非人類來源之FR或CDR殘基的完整人類抗體。 (v) 抗體片段 抗體片段可藉由傳統方式(諸如酶促消化)或藉由重組技術產生。在某些情形下，使用抗體片段而非使用整個抗體具有優勢。較小尺寸之片段允許迅速清除且可導致對進入實體腫瘤之改良。某些抗體片段之評論參見Hudson等人，(2003)Nat. Med. 9:129-134。 已開發出多種技術用於產生抗體片段。傳統上，此等片段可經由完整抗體之蛋白水解消化獲得(參見例如Morimoto等人，Journal of Biochemical and Biophysical Methods 24:107-117 (1992)；及Brennan等人，Science , 229:81 (1985))。然而，此等片段現在可由重組宿主細胞直接產生。Fab、Fv及ScFv抗體片段均可表現於大腸桿菌中且自大腸桿菌分泌，因此允許容易地產生大量此等片段。可自上文所討論之抗體噬菌體文庫中分離抗體片段。或者，可直接自大腸桿菌中回收Fab'-SH片段且使其化學偶合形成F(ab')2 片段(Carter等人，Bio/Technology 10:163-167 (1992))。根據另一方法，F(ab')2 片段可直接自重組宿主細胞培養物中分離。活體內半衰期延長且包含救助受體結合抗原決定基殘基之Fab及F(ab')2 片段描述於美國專利第5,869,046號中。熟練從業者將顯而易知抗體片段之其他製造技術。在某些實施例中，抗體為單鏈Fv片段(scFv)。參見WO 93/16185、美國專利第5,571,894號及第5,587,458號。Fv及scFv為具有不含恆定區之完整結合位點的僅有物質；因此，其可適於在活體內使用期間減少非特異性結合。可構築scFv融合蛋白以使效應蛋白融合於scFv之胺基或羧基末端。參見Antibody Engineering , Borrebaeck編，同上文。抗體片段亦可為例如美國專利第5,641,870號中所述之「線性抗體」。此類線性抗體可為單特異性或雙特異性抗體。 (vi) 多特異性抗體 多特異性抗體對至少兩個不同抗原決定基具有結合特異性，其中抗原決定基通常來自不同抗原。雖然此類分子通常僅結合兩個不同抗原決定基(亦即雙特異性抗體，BsAb)，但具有額外特異性之抗體(諸如三特異性抗體)當用於本文中時係由此表達所涵蓋。可製備全長抗體或抗體片段形式之雙特異性抗體(例如F(ab')2 雙特異性抗體)。 製備雙特異性抗體之方法為此項技術所已知。傳統上全長雙特異性抗體之產生係基於兩個免疫球蛋白重鏈-輕鏈對之共表現，其中兩條鏈具有不同特異性(Millstein等人，Nature , 305:537-539 (1983))。由於免疫球蛋白重鏈及輕鏈之隨機分配，此等融合瘤(四源融合瘤)產生10種不同抗體分子之潛在混合物，其中僅一種具有正確雙特異性結構。一般藉由親和力層析步驟進行之正確分子之純化相當麻煩，且產量低。類似程序揭示於WO 93/08829中及Traunecker等人，EMBO J. , 10:3655-3659 (1991)中。 根據不同方法，將具所需結合特異性之抗體可變域(抗體-抗原結合位點)融合至免疫球蛋白恆定域序列。融合較佳係與包含至少部分鉸鏈區、CH2及CH3區之免疫球蛋白重鏈恆定域之融合。通常使存在於至少一個該等融合中之第一重鏈恆定區(CH1)含有用於結合輕鏈之必要位點。將編碼免疫球蛋白重鏈融合體及(若需要)免疫球蛋白輕鏈之DNA***單獨表現載體中，且將其共轉染至適合宿主生物體中。當在構築中所使用之三個多肽鏈的不等比率提供最佳產率時，此提供在實施例中調整三個多肽片段之相互比例的極大靈活性。然而，當等比率之至少兩個多肽鏈的表現造成高產率或當比率並非特別重要時，可能將兩個或全部三個多肽鏈之編碼序列***一個表現載體中。 在此方法之一個實施例中，雙特異性抗體由在一個臂中具有第一結合特異性之雜交免疫球蛋白重鏈與在另一臂中之雜交免疫球蛋白重鏈-輕鏈對(提供第二結合特異性)組成。已發現由於免疫球蛋白輕鏈僅存在於一半雙特異性分子中提供一種簡便之分離方式，故此不對稱結構促進所需雙特異性化合物與非所要免疫球蛋白鏈組合之分離。此方法揭示於WO 94/04690中。關於產生雙特異性抗體之其他細節參見例如Suresh等人，Methods in Enzymology , 121:210 (1986)。 根據描述於WO96/27011中之另一方法，可工程設計一對抗體分子間之界面以使由重組細胞培養物回收的雜二聚體之百分比最大化。一個界面包含抗體恆定域之CH 3結構域之至少一部分。以此方法，使第一抗體分子界面之一或多條小胺基酸側鏈經較大側鏈(例如酪胺酸或色胺酸)置換。尺寸與大側鏈相同或類似之互補「腔」藉由將大胺基酸側鏈置換為較小胺基酸側鏈(例如丙胺酸或蘇胺酸)而形成於第二抗體分子界面上。此提供使雜二聚體之產率增加超過其他不合需要之最終產物(諸如均二聚體)的機制。 雙特異性抗體包括交聯或「異源結合」抗體。舉例而言，呈異源結合物形式之抗體中之一者可與抗生物素蛋白偶合，另一者與生物素偶合。舉例而言，已提出此類抗體使免疫系統細胞靶向非所要細胞(美國專利第4,676,980號)，且用於治療HIV感染(WO 91/00360、WO 92/200373及EP 03089)。異源結合抗體可使用任何便利的交聯方法製備。適合交聯劑以及許多交聯技術在此項技術中為熟知的且揭示於美國專利第4,676,980號中。 文獻中亦已描述自抗體片段產生雙特異性抗體之技術。舉例而言，可使用化學鍵聯製備雙特異性抗體。Brennan等人，Science , 229:81 (1985)描述一種其中完整抗體經蛋白質裂解可產生F(ab')2 片段的程序。在二硫醇錯合劑亞砷酸鈉存在下還原此等片段以穩定鄰近二硫醇且防止分子間二硫鍵形成。接著將所產生之Fab'片段轉化為硫代硝基苯甲酸酯(TNB)衍生物。接著藉由用巰基乙胺還原將一種Fab'-TNB衍生物再轉化為Fab'-硫醇，且將其與等莫耳量之另一Fab'-TNB衍生物混合以形成雙特異性抗體。所產生之雙特異性抗體可用作用於選擇性固定酶之試劑。 近期發展已促進自大腸桿菌中直接回收Fab'-SH片段，該等片段可經化學偶合形成雙特異性抗體。Shalaby等人，J. Exp. Med. , 175:217-225 (1992)描述了完全人類化雙特異性抗體F(ab')2 分子之製備。各Fab'片段分別自大腸桿菌中分泌且經受活體外定向化學偶合以形成雙特異性抗體。 亦已描述多種直接由重組細胞培養物製備及分離雙特異性抗體片段之技術。舉例而言，已使用白胺酸拉鏈產生雙特異性抗體。Kostelny等人，J. Immunol., 148 (5):1547-1553 (1992)。藉由基因融合將來自Fos及Jun蛋白質之白胺酸拉鏈肽連接至兩種不同抗體之Fab'部分。在鉸鏈區還原抗體均二聚體以形成單體，且接著使其再氧化以形成抗體雜二聚體。此方法亦可用於產生抗體均二聚體。Hollinger等人，Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)所述之「雙功能抗體」技術為製備雙特異性抗體片段提供了替代機制。該等片段包含經連接子連接至輕鏈可變域(VL )之重鏈可變域(VH )，該連接子過短而使相同鏈上之兩個結構域之間無法配對。因此，迫使一個片段之VH 及VL 域與另一片段之互補VL 及VH 域配對，藉此形成兩個抗原結合位點。亦已報導另一種藉由使用單鏈Fv(sFv)二聚體來製備雙特異性抗體片段之策略。參見Gruber等人，J. Immunol. , 152:5368 (1994)。 具有兩價以上的抗體亦考慮在內。舉例而言，可製備三特異性抗體。Tuft 等人. J. Immunol. 147: 60 (1991)。 (vii) 單結構域抗體 在一些實施例中，本發明之抗體為單結構域抗體。單結構域抗體為包含抗體之全部或一部分重鏈可變域或者全部或一部分輕鏈可變域之單一多肽鏈。在某些實施例中，單結構域抗體為人類單結構域抗體(Domantis, Inc., Waltham, Mass.；參見例如美國專利第6,248,516 B1號)。在一個實施例中，單結構域抗體由抗體之全部或一部分重鏈可變域組成。 (viii)抗體變異體 在一些實施例中，涵蓋本文所述之抗體的胺基酸序列修飾。舉例而言，可需要改良抗體之結合親和力及/或其他生物學特性。可藉由將適當變化引入編碼抗體之核苷酸序列中或藉由肽合成來製備抗體之胺基酸序列變異體。此類修飾包括例如抗體胺基酸序列內之殘基缺失及/或***及/或取代。可進行缺失、***及取代之任何組合以獲得最終構築體，其限制條件為最終構築體具有所需之特徵。可在製造序列時將胺基酸變化引入標的抗體胺基酸序列中。 (ix) 抗體衍生物 本發明之抗體可進一步經修飾以含有此項技術中已知且容易獲得之額外非蛋白部分。在某些實施例中，適於衍生化抗體之部分為水溶性聚合物。水溶性聚合物之非限制性實例包括(但不限於)聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纖維素、葡聚糖、聚乙烯醇、聚乙烯吡咯啶酮、聚-1,3-二氧雜環戊烷、聚-1,3,6-三噁烷、乙烯/順丁烯二酸酐共聚物、聚胺基酸(均聚物或無規共聚物)及葡聚糖或聚(n-乙烯吡咯啶酮)聚乙二醇、丙二醇均聚物、聚氧化丙烯/氧化乙烯共聚物、聚氧乙烯多元醇(例如甘油)、聚乙烯醇及其混合物。聚乙二醇丙醛因其於水中之穩定性而可於製造時具有優點。聚合物可具有任何分子量，且可分支或未分支。連接至抗體之聚合物數目可變化，且若連接一個以上聚合物，則該等聚合物可為相同或不同分子。一般而言，用於衍生作用之聚合物之數目及/或類型可基於包括(但不限於)待改良抗體之特殊特性或功能，抗體衍生物是否將用於指定條件下之療法等考慮來確定。 (x) 載體、宿主細胞及重組方法 亦可使用重組方法產生抗體。為重組產生抗-抗原抗體，將編碼該抗體之核酸分離且***可複製載體中以便進一步選殖(擴增DNA)或以便表現。可使用習知程序(例如藉由使用能夠特異性結合編碼抗體重鏈及輕鏈之基因之寡核苷酸探針)容易地分離及定序編碼抗體之DNA。可利用多種載體。載體組分一般包括(但不限於)以下中之一或多者：信號序列、複製起點、一或多個標記基因、強化子成分、啟動子及轉錄終止序列。 (a)信號序列組分 本發明之抗體不僅可直接重組產生，而且還可以與異源多肽融合之融合多肽的形式產生，該異源多肽較佳為信號序列或在成熟蛋白質或多肽之N端處具有特定裂解位點之其他多肽。所選擇之異源信號序列較佳為可由宿主細胞識別及加工(亦即由信號肽酶裂解)之信號序列。對於不識別及加工原生抗體信號序列之原核宿主細胞，將該信號序列取代為例如選自鹼性磷酸酶、青黴素酶、lpp或熱穩定性腸毒素II前導序列之群的原核信號序列。對於酵母分泌，原生信號序列可取代為例如酵母轉化酶前導序列、因子前導序列(包括酵母菌(Saccharomyces )及克魯維酵母(Kluyveromyces )α-因子前導序列)或酸性磷酸酶前導序列、白色念珠菌(C. albicans )葡糖澱粉酶前導序列或WO 90/13646中所述之信號。在哺乳動物細胞表現中，可使用哺乳動物信號序列以及病毒分泌前導序列(例如單純疱疹gD信號)。 (b) 複製起點 表現與選殖載體皆含有使載體能夠在一或多種所選宿主細胞中複製的核酸序列。通常，在選殖載體中，此序列為使載體能夠不依賴於宿主染色體DNA複製的序列，且包括複製起點或自主複製序列。熟知用於多種細菌、酵母及病毒之此類序列。來自質體pBR322之複製起點適於大部分革蘭氏陰性細菌(Gram-negative bacteria)，2μ質體起點適於酵母，且各種病毒起點(SV40、多瘤病毒、腺病毒、VSV或BPV)適用於在哺乳動物細胞中選殖載體。一般而言，哺乳動物表現載體無需複製起點組份(因SV40起點含有早期啟動子，故通常可僅使用該起點)。 (c) 選擇基因組分 表現及選殖載體可含有選擇基因，亦稱為可選標記物。典型選擇基因編碼以下蛋白質：(a)賦予對抗生素或其他毒素(例如安比西林(ampicillin)、新黴素(neomycin)、甲胺喋呤(methotrexate)或四環素(tetracycline))之耐藥性，(b)補充營養缺陷缺乏，或(c)供應不可自複雜培養基中獲得之關鍵營養，例如供給桿菌編碼D-丙胺酸消旋酶之基因。 選擇流程之一個實例利用藥物使宿主細胞之生長停滯。經異源基因成功轉形之彼等細胞產生呈現耐藥性之蛋白質且因此在選擇方案中存活。此類顯性選擇之實例使用藥物新黴素、黴酚酸及濕黴素(hygromycin)。 哺乳動物細胞之適合可選標記物之另一實例為使得能夠鑑別有能力接納編碼抗體之核酸之細胞的彼等標記物，諸如DHFR、麩醯胺酸合成酶(GS)、胸苷激酶、金屬硫蛋白-I及金屬硫蛋白-II(較佳為靈長類金屬硫蛋白基因)、腺苷脫胺酶、鳥胺酸脫羧酶等。 舉例而言，藉由在含有甲胺喋呤(Mtx)(DHFR之競爭性拮抗劑)之培養基中培養經DHFR基因轉形之細胞來鑑別轉形體。在此等條件下，DHFR基因與任何其他共轉形核酸一起擴增。可使用缺乏內源性DHFR活性之中國倉鼠卵巢(CHO)細胞株(例如ATCC CRL-9096)。 或者，藉由在含有L-甲硫胺酸磺醯亞胺(Msx)(GS之抑制劑)之培養基中培養轉形體來鑑別經GS基因轉形之細胞。在此等條件下，GS基因與任何其他共轉形核酸一起擴增。GS選擇/擴增系統可與如上所述之DHFR選擇/擴增系統組合使用。 或者，可藉由在含有針對可選標記物之選擇劑(諸如胺基糖苷抗生素，例如康黴素(kanamycin)、新黴素或G418)的培養基中進行細胞生長來選擇經編碼所關注抗體之DNA序列、野生型DHFR基因及另一可選標記物(諸如胺基糖苷3'-磷酸轉移酶(APH))轉形或共轉形之宿主細胞(尤其是含有內源DHFR之野生型宿主)。參見美國專利第4,965,199號。 適用於酵母中之選擇基因為存在於酵母質體YRp7中之trp 1基因(Stinchcomb等人，Nature , 282:39 (1979))。trp 1基因為不能在色胺酸中生長的突變型酵母菌株(例如ATCC編號44076或PEP4-1)提供選擇標記。Jones,Genetics , 85:12 (1977)。隨後酵母宿主細胞基因組中trp 1機能障礙之存在為藉由在無色胺酸存在情況下培養來偵測轉形提供有效環境。類似地，Leu 2缺陷型酵母菌株(ATCC 20,622或38,626)由攜帶Leu 2基因之已知質體補充。 另外，源自1.6 μm環狀質體pKD1之載體可用於轉形克魯維酵母。或者，針對乳酸克魯維酵母報導了用於大規模製造重組牛凝乳酶之表現系統。Van den Berg,Bio/Technology , 8:135 (1990)。亦已揭示用於利用工業克魯維酵母菌株分泌成熟重組人類血清白蛋白的穩定多複本表現載體。Fleer等人，Bio/Technology , 9:968-975 (1991)。 (d) 啟動子組分 表現及選殖載體通常含有由宿主生物體識別且可操作地連接於編碼抗體之核酸的啟動子。適用於原核宿主之啟動子包括pho A啟動子、β-內醯胺酶及乳糖啟動子系統、鹼性磷酸酶啟動子、色胺酸(trp)啟動子系統及雜合啟動子(諸如tac啟動子)。然而，其他已知之細菌啟動子亦適合。用於細菌系統中之啟動子亦將含有可操作地連接於編碼抗體之DNA的夏因-達爾加諾(Shine-Dalgarno；S.D.)序列。 已知用於真核生物之啟動子序列。實際上所有真核基因均具有位於轉錄起始位點上游大約25至30個鹼基處之AT增濃區。發現於多種基因之轉錄起始處上游70至80個鹼基處的另一序列為CNCAAT區，其中N可為任何核苷酸。大部分真核基因的3'端處為AATAAA序列，該序列可為編碼序列之3'端增加聚A尾的信號。所有此等序列均適於***至真核表現載體中。 適用於酵母宿主之啟動子序列之實例包括3-磷酸甘油酸激酶或其他醣解酶之啟動子，諸如烯醇酶、甘油醛-3-磷酸脫氫酶、己醣激酶、丙酮酸脫羧酶、磷酸果糖激酶、葡萄糖-6-磷酸異構酶、3-磷酸甘油酸歧化酶、丙酮酸激酶、磷酸丙醣異構酶、磷酸葡糖異構酶及葡萄糖激酶。 具有轉錄受生長條件控制之其他優點之誘導型啟動子的其他酵母啟動子為用於以下之啟動子區：醇去氫酶2、異細胞色素C、酸性磷酸酶、與氮代謝相關之分解酶、金屬硫蛋白、甘油醛-3-磷酸去氫酶及負責麥芽糖及半乳糖利用之酶。EP 73,657中進一步描述適用於酵母表現之載體及啟動子。酵母強化子亦宜與酵母啟動子一起使用。 可例如藉由自以下獲得之啟動子控制哺乳動物宿主細胞中自載體進行抗體轉錄：諸如多形瘤病毒、雞痘病毒、腺病毒(諸如腺病毒2)、牛乳頭狀瘤病毒、鳥肉瘤病毒、細胞巨大病毒、反轉錄病毒、B型肝炎病毒、猿猴病毒40(SV40)之病毒的基因組；或異源哺乳動物啟動子(例如肌動蛋白啟動子或免疫球蛋白啟動子)；熱休克啟動子，其限制條件為該等啟動子與宿主細胞系統相容。 便利地獲得SV40病毒之早期及晚期啟動子，作為亦含有SV40病毒複製起點之SV40限制片段。便利地獲得人類巨細胞病毒之立即早期啟動子，作為HindIII E限制片段。在哺乳動物宿主中使用牛乳頭狀瘤病毒作為載體表現DNA之系統揭示於美國專利第4,419,446號中。此系統之修飾描述於美國專利第4,601,978號中。人類β-干擾素cDNA在來自單純性疱疹病毒之胸苷激酶啟動子控制下於小鼠細胞中之表現亦參見Reyes等人，Nature 297:598-601 (1982)。或者，可使用勞斯肉瘤病毒(Rous Sarcoma Virus)之長末端重複序列作為啟動子。 (e) 強化子要素組分 通常藉由將強化子序列***載體中來增加編碼本發明抗體之DNA由高級真核生物之轉錄。現已知多種來自哺乳動物基因(血球蛋白、彈性蛋白酶、白蛋白、α-胎蛋白及胰島素)之強化子序列。然而，吾人通常使用來自真核細胞病毒之強化子。實例包括複製起點後側之SV40強化子(bp 100-270)、巨細胞病毒早期啟動子強化子、複製起點後側之多瘤病毒強化子及腺病毒強化子。關於用於活化真核啟動子之增強元件亦可參見Yaniv,Nature 297:17-18 (1982)。強化子可拼接至載體中抗體編碼序列之5'或3'位置處，但較佳位於啟動子之5'位點。 (f) 轉錄終止組分 用於真核宿主細胞(酵母、真菌、昆蟲、植物、動物、人類或來自其他多細胞生物體之有核細胞)之表現載體亦含有終止轉錄及穩定mRNA所必需的序列。此類序列通常可自真核或病毒DNA或cDNA之5'且有時3'非轉譯區獲得。此等區域含有在編碼抗體之mRNA之未轉譯部分中以聚腺苷酸化片段形式轉錄的核苷酸區段。一種適用之轉錄終止組份為牛生長激素多聚腺嘌呤化區。參見WO94/11026及其中所揭示之表現載體。 (g) 宿主細胞之選擇及轉形 適用於在本文之載體中選殖或表現DNA之宿主細胞為如上文所述之原核生物、酵母或高級真核生物細胞。適於此目的之原核生物包括真細菌，諸如革蘭氏陰性或革蘭氏陽性生物體，例如腸內菌科(Enterobacteriaceae)，諸如埃希氏菌屬(Escherichia )(例如大腸桿菌)、腸內菌屬(Enterobacter )、歐文氏菌屬(Erwinia )、克雷伯氏菌屬(Klebsiella )、變形桿菌屬(Proteus )、沙門氏菌屬(Salmonella )(例如鼠傷寒沙門氏菌(Salmonella typhimurium ))、沙雷氏菌屬(Serratia )(例如黏質沙雷氏菌(Serratia marcescans ))及志賀氏菌屬(Shigella )以及芽孢桿菌屬(Bacilli )(諸如枯草芽孢桿菌(B. subtilis )及地衣芽孢桿菌(B. licheniformis )(例如1989年4月12日公開之DD 266,710中揭示之地衣芽孢桿菌41P))、假單胞菌屬(Pseudomonas )(諸如綠膿假單胞菌(P. aeruginosa ))及鏈黴菌屬(Streptomyces )。儘管諸如大腸桿菌B、大腸桿菌X1776(ATCC 31,537)及大腸桿菌W3110(ATCC 27,325)之其他品系亦為適合的，但一種較佳大腸桿菌選殖宿主為大腸桿菌294(ATCC 31,446)。此等實例為說明性的而非限制性的。 可在細菌中產生全長抗體、抗體融合蛋白及抗體片段，尤其當不需要糖基化及Fc效應功能時，諸如當使治療抗體與自身展示破壞腫瘤細胞之功效之細胞毒性劑(例如毒素)結合時。全長抗體在循環中具有較長半衰期。在大腸桿菌中產生更快且更節省成本。抗體片段及多肽於細菌中之表現參見例如，美國專利第5,648,237號(Carter等人)、美國專利第5,789,199號(Joly等人)、美國專利第5,840,523號(Simmons等人)，其描述使表現及分泌最佳之轉譯起始區(TIR)及信號序列。亦參見Charlton,Methods in Molecular Biology , 第248卷 (B. K. C. Lo編, Humana Press, Totowa, N.J., 2003), 第245頁-第254頁，描述抗體片段於大腸桿菌中之表現。表現後，可溶性部分中之抗體可與大腸桿菌細胞糊狀物分離且可視同型而定藉由例如蛋白質A或G管柱純化。可類似於純化例如CHO細胞中表現之抗體之方法來進行最終純化。 除原核生物外，諸如絲狀真菌或酵母之真核微生物為編碼抗體之載體的適合選殖或表現宿主。在低級真核宿主微生物中最常使用釀酒酵母(Saccharomyces cerevisiae )或普通麵包酵母(baker's yeast)。然而，許多其他屬、種及病毒株通常可用且適用於此處，諸如粟酒裂殖酵母(Schizosaccharomyces pombe )；克魯維酵母宿主，諸如乳酸克魯維酵母、脆壁克魯維酵母(K. fragilis )(ATCC 12,424)、保加利亞克魯維酵母(K. bulgaricus )(ATCC 16,045)、威克克魯維酵母(K. wickeramii )(ATCC 24,178)、克魯維雄酵母(K. waltii )(ATCC 56,500)、果妮克魯維酵母(K. drosophilarum )(ATCC 36,906)、耐熱克魯維酵母(K. thermotolerans )及馬克斯克魯維酵母(K. marxianus )；耶氏酵母屬(yarrowia )(EP 402,226)；甲醇酵母(Pichia pastoris )(EP 183,070)；假絲酵母(Candida )；里氏木黴(Trichoderma reesia )(EP 244,234)；粗厚神經胞子菌(Neurospora crassa )；施氏酵母(Schwanniomyces )，諸如許旺酵母(Schwanniomyces occidentalis )；及絲狀真菌，諸如紅黴菌屬(Neurospora )、青黴菌屬(Penicillium )、彎頸黴屬(Tolypocladium )及麴菌屬(Aspergillus )宿主，諸如構巢麯黴(A. nidulans )及黑麯黴(A. niger )。論述酵母及絲狀真菌用於製造治療蛋白質之用途的評論參見例如Gerngross,Nat. Biotech. 22:1409-1414 (2004)。 可選擇某些真菌及酵母菌株，其中糖基化路徑已經「人類化」，導致產生具有部分或完全人類糖基化模式之抗體。參見例如Li等人，Nat. Biotech. 24:210-215 (2006) (描述甲醇酵母中糖基化路徑之人類化)；及Gerngross等人，同上文。 適於表現糖基化抗體之宿主細胞亦可自多細胞生物體(無脊椎動物及脊椎動物)獲得。無脊椎動物細胞之實例包括植物及昆蟲細胞。已鑑別多種桿狀病毒病毒株及變異體以及來自諸如草地黏蟲(Spodoptera frugiperda )(毛蟲)、埃及伊蚊(Aedes aegypti )(蚊子)、白紋伊蚊(Aedes albopictus )(蚊子)、黑腹果蠅(Drosophila melanogaster )(果蠅)及家蠶(Bombyx mori )之宿主的相應許可昆蟲宿主細胞。用於轉染之多種病毒株公開可得，例如苜蓿丫紋夜蛾(Autographa californica ) NPV之L-1變異體及家蠶NPV之Bm-5病毒株，且根據本發明之該等病毒可用作本文中之病毒，尤其用於轉染草地黏蟲細胞。 棉花、玉米、馬鈴薯、大豆、矮牽牛、番茄、浮萍(浮萍科(Lemnaceae ))、紫苜蓿(蒺藜苜蓿(M. truncatula ))及菸草之植物細胞培養物亦可用作宿主。參見例如美國專利第5,959,177號、第6,040,498號、第6,420,548號、第7,125,978號及第6,417,429號(描述用於在專殖基因植物中產生抗體之PLANTIBODIESTM 技術)。 脊椎動物細胞可用作宿主，且在培養物(組織培養物)中使脊椎動物細胞增殖已成為常規程序。適用哺乳動物宿主細胞株之實例為經SV40轉形之猴腎CV1細胞株(COS-7，ATCC CRL 1651)；人類胚腎細胞株(293細胞或經次選殖以供在懸浮培養物中生長之293細胞，Graham等人，J. Gen Virol. 36:59 36:59 (1977))；幼倉鼠腎細胞(BHK, ATCC CCL 10)；小鼠支持細胞(TM4, Mather,Biol. Reprod. 23:243-251 (1980))；猴腎細胞(CV1 ATCC CCL 70)；非洲綠猴腎細胞(African green monkey kidney cells)(VERO-76, ATCC CRL-1587)；人類子宮頸癌細胞(HELA, ATCC CCL 2)；犬腎細胞(MDCK, ATCC CCL 34)；布法羅大鼠肝細胞(buffalo rat liver cell)(BRL 3A, ATCC CRL 1442)；人類肺細胞(W138, ATCC CCL 75)；人類肝細胞(Hep G2, HB 8065)；小鼠***腫瘤(MMT 060562, ATCC CCL51)；TRI細胞(Mather等人，Annals N.Y. Acad. Sci. 383:44-68 (1982))；MRC 5細胞；FS4細胞；及人類肝癌細胞株(Hep G2)。其他適用哺乳動物宿主細胞株包括中國倉鼠卵巢(CHO)細胞，包括DHFR- CHO細胞(Urlaub等人，Proc. Natl. Acad. Sci. USA 77:4216 (1980))；及骨髓瘤細胞株，諸如NS0及Sp2/0。適於抗體產生之某些哺乳動物宿主細胞株之評論參見例如Yazaki及Wu,Methods in Molecular Biology , 第248卷(B. K. C. Lo編, Humana Press, Totowa, N.J., 2003), 第255頁-第268頁。 用上述用於產生抗體之表現或選殖載體轉形宿主細胞，且將其培養於經改質以適於誘導啟動子、選擇轉形體或擴增編碼所需序列之基因的習知營養培養基中。 (h) 培養宿主細胞 可將用於產生本發明抗體之宿主細胞培養於多種培養基中。市售培養基，諸如漢氏F10(Ham's F10，Sigma)、最低必需培養基(MEM)(Sigma)、RPMI-1640(Sigma)及杜貝可氏改良型伊格爾氏培養基(Dulbecco's Modified Eagle's Medium，DMEM)(Sigma)，適用於培養宿主細胞。此外，以下文獻中所述之任一種培養基可用作該等宿主細胞之培養基：Ham等人，Meth. Enz. 58:44 (1979)；Barnes等人，Anal. Biochem. 102:255 (1980)；美國專利第4,767,704號；第4,657,866號；第4,927,762號；第4,560,655號；或第5,122,469號；WO 90/03430；WO 87/00195；或美國專利Re.30,985。任何此等培養基均可視需要補充激素及/或其他生長因子(諸如胰島素、轉鐵蛋白或表皮生長因子)、鹽(諸如氯化鈉、鈣、鎂及磷酸鹽)、緩衝液(諸如HEPES)、核苷酸(諸如腺苷及胸苷)、抗生素(諸如GENTAMYCINTM 藥物)、痕量元素(定義為無機化合物，通常以在微莫耳範圍內之最終濃度存在)及葡萄糖或等效能源。亦可包括熟習此項技術者已知之適當濃度的任何其他必需補充劑。培養條件(諸如溫度、pH及其類似條件)為先前選擇進行表現之宿主細胞所用的條件，且為一般技術者顯而易知。 (xi) 抗體純化 當使用重組技術時，抗體可於細胞內、周質間隙中產生或直接分泌至培養基中。若抗體於細胞內產生，則作為第一步驟，例如藉由離心或超濾移除宿主細胞或已溶解片段之顆粒碎片。Carter等人，Bio/Technology 10:163-167 (1992)描述用於分離分泌至大腸桿菌之周質空間中的抗體之程序。簡言之，細胞糊狀物在乙酸鈉(pH 3.5)、EDTA及苯基甲基磺醯氟(PMSF)存在下經約30分鐘解凍。可藉由離心移除細胞碎片。在抗體分泌至培養基中之情形下，通常首先使用市售蛋白質濃縮過濾器(例如Amicon或Millipore Pellicon超濾單元)濃縮此等表現系統之清液層。在任何先前步驟中可包括諸如PMSF之蛋白酶抑制劑以抑制蛋白水解，且可包括抗生素以防止外來污染物生長。 可使用例如羥磷灰石層析、疏水相互作用層析、凝膠電泳、透析及親和力層析來純化自細胞製備之抗體組合物，且親和力層析為其中一種通常較佳之純化步驟。蛋白質A作為親和力配位體之適用性視物種及存在於抗體中之任何免疫球蛋白Fc域的同型而定。蛋白質A可用於純化基於人類γ1、γ2或γ4重鏈之抗體(Lindmark等人，J. Immunol. Meth. 62:1-13 (1983))。蛋白質G推薦用於所有小鼠同型及人類γ3(Guss等人，EMBO J. 5:15671575 (1986))。雖然親和力配位體所連接之基質最常為瓊脂糖，但可利用其他基質。與用瓊脂糖可實現之流動速率及處理時間相比，機械穩定性基質(諸如可控孔度玻璃或聚(苯乙烯二乙烯基)苯)流動速率更快且處理時間更短。當抗體包含CH 3域時，Bakerbond ABXTM 樹脂(J. T. Baker；Phillipsburg, N.J.)適用於純化。亦可視待回收抗體而定利用其他用於蛋白質純化之技術，諸如離子交換柱分餾法、乙醇沈積法、逆相HPLC、二氧化矽層析、肝素SEPHAROSE™層析、陰離子或陽離子交換樹脂(諸如聚天冬胺酸管柱)層析、層析聚焦、SDS-PAGE及硫酸銨沈澱。 一般而言，用於製備供研究、測試及臨床使用之抗體的各種方法在此項技術中已充分確立，與上述方法一致且/或被熟習此項技術者視為適用於所關注之特定抗體。C. 選擇生物活性抗體 可對如上文所述產生之抗體進行一或多種「生物活性」分析法來從治療觀點選擇具有有益特性之抗體或選擇保留抗體之生物活性的調配物及條件。可測試抗體與使其升高之抗原結合之能力。舉例而言，對於抗PDL1抗體，可在偵測與PDL1結合之能力的分析法中評估抗體之抗原結合特性。在一些實施例中，可藉由飽和結合；ELISA；及/或競爭分析(例如RIA's)來測定抗體之結合。同樣，可對抗體進行其他生物活性分析，例如以評估其作為治療劑之有效性。此類分析法在此項技術中已知且視目標抗原及抗體之預期用途而定。舉例而言，抗體阻斷之PD-L1之生物作用可在CD8+T細胞、淋巴球性脈絡叢腦膜炎病毒(LCMV)小鼠模型及/或同系腫瘤模型(例如US專利8,217,149中所描述)中評定。 為了篩選結合於所關注抗原上之特定抗原決定基的抗體(例如阻斷實例之抗PDL1抗體與PD-L1結合者)，可進行常規交叉阻斷分析法(諸如Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)中所述者。或者，可執行例如如Champe等人，J. Biol. Chem. 270:1388-1394 (1995)中所述之抗原決定基定位來判定抗體是否結合相關抗原決定基。D. 製備調配物 在製備相關抗體(例如用於製造可如本文所揭示經調配之抗體的技術將在下文中詳述且在此項技術中已知)後，製備包含其之醫藥調配物。在某些實施例中，待調配之抗體未進行預先凍乾且本文中之相關調配物為水性調配物。在某些實施例中，抗體為全長抗體。在一個實施例中，調配物中之抗體為抗體片段，諸如F(ab')2 ，可能需要解決在該情況下全長抗體不會出現之問題(諸如抗體剪接成Fab)。調配物中存在之抗體的治療有效量係藉由考慮例如所需劑量體積及投與模式來測定。約25 mg/mL至約150 mg/mL，或約30 mg/mL至約140 mg/mL，或約35 mg/mL至約130 mg/mL，或約40 mg/mL至約120 mg/mL，或約50 mg/mL至約130 mg/mL，或約50 mg/mL至約125 mg/mL，或約50 mg/mL至約120 mg/mL，或約50 mg/mL至約110 mg/mL，或約50 mg/mL至約100 mg/mL，或約50 mg/mL至約90 mg/mL，或約50 mg/mL至約80 mg/mL，或約54 mg/mL至約66 mg/mL為調配物中之例示性抗體濃度。 製備包含在pH緩衝溶液中之抗體的水性調配物。本發明之緩衝液具有約5.0至約7.0範圍內之pH。在某些實施例中，pH在約5.0至約6.5範圍內，pH在約5.0至約6.4範圍內，在約5.0至約6.3範圍內，pH在約5.0至約6.2範圍內，pH在約5.0至約6.1範圍內，pH在約5.5至約6.1範圍內，pH在約5.0至約6.0範圍內，pH在約5.0至約5.9範圍內，pH在約5.0至約5.8範圍內，pH在約5.1至約6.0範圍內，pH在約5.2至約6.0範圍內，pH在約5.3至約6.0範圍內，pH在約5.4至約6.0範圍內，pH在約5.5至約6.0範圍內，pH在約5.6至約6.0範圍內，pH在約5.7至約6.0範圍內，或pH在約5.8至約6.0範圍內。在本發明之某些實施例中，調配物具有6.0或約6.0之pH。在本發明之某些實施例中，調配物具有5.9或約5.9之pH。在本發明之某些實施例中，調配物具有5.8或約5.8之pH。在本發明之某些實施例中，調配物具有5.7或約5.7之pH。在本發明之某些實施例中，調配物具有5.6或約5.6之pH。在本發明之某些實施例中，調配物具有5.5或約5.5之pH。在本發明之某些實施例中，調配物具有5.4或約5.4之pH。在本發明之某些實施例中，調配物具有5.3或約5.3之pH。在本發明之某些實施例中，調配物具有5.2或約5.2之pH。將pH控制於此範圍中之緩衝液之實例包括組胺酸(諸如L-組胺酸)或乙酸鈉。在某些實施例中，緩衝液含有濃度為約15 mM至約25 mM之組胺酸乙酸鹽或乙酸鈉。在本發明之某些實施例中，緩衝液含有濃度為約15 mM至約25 mM、約16 mM至約25 mM、約17 mM至約25 mM、約18 mM至約25 mM、約19 mM至約25 mM、約20 mM至約25 mM、約21 mM至約25 mM、約22 mM至約25 mM、約15 mM、約16 mM、約17 mM、約18 mM、約19 mM、約20 mM、約21 mM、約22 mM、約23 mM、約24 mM或約25 mM之組胺酸乙酸鹽或乙酸鈉。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.0。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.1。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.2。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.3。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.4。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.5。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.6。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.7。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.8。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.9。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.0。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.1。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.2。在一個實施例中，緩衝液為約20 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.3。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.2。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.3。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.4。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.5。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.6。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.7。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.8。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 5.9。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.0。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.1。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.2。在一個實施例中，緩衝液為約25 mM之量的組胺酸乙酸鹽或乙酸鈉，pH 6.3。 調配物進一步包含約60 mM至約240 mM之量的蔗糖。在一些實施例中，調配物中之蔗糖為約60 mM至約230 mM、約60 mM至約220 mM、約60 mM至約210 mM、約60 mM至約200 mM、約60 mM至約190 mM、約60 mM至約180 mM、約60 mM至約170 mM、約60 mM至約160 mM、約60 mM至約150 mM、約60 mM至約140 mM、約80 mM至約240 mM、約90 mM至約240 mM、約100 mM至約240 mM、約110 mM至約240 mM、約120 mM至約240 mM、約130 mM至約240 mM、約140 mM至約240 mM、約150 mM至約240 mM、約160 mM至約240 mM、約170 mM至約240 mM、約180 mM至約240 mM、約190 mM至約240 mM、約200 mM至約240 mM、約80 mM至約160 mM、約100 mM至約140 mM，或約110 mM至約130 mM。在一些實施例中，調配物中之蔗糖為約60 mM、約70 mM、約80 mM、約90 mM、約100 mM、約110 mM、約120 mM、約130 mM、約140 mM、約150 mM、約160 mM、約170 mM、約180 mM、約190 mM、約200 mM、約210 mM、約220 mM、約230 mM或約240 mM。 在一些實施例中，調配物中之抗體濃度為約40 mg/ml至約125 mg/ml。在一些實施例中，調配物中之抗體濃度為約40 mg/ml至約120 mg/ml、約40 mg/ml至約110 mg/ml、約40 mg/ml至約100 mg/ml、約40 mg/ml至約90 mg/ml、約40 mg/ml至約80 mg/ml、約40 mg/ml至約70 mg/ml、約50 mg/ml至約120 mg/ml、約60 mg/ml至約120 mg/ml、約70 mg/ml至約120 mg/ml、約80 mg/ml至約120 mg/ml、約90 mg/ml至約120 mg/ml，或約100 mg/ml至約120 mg/ml。在一些實施例中，調配物中之抗體濃度為約60 mg/ml。在一些實施例中，調配物中之抗體濃度為約65 mg/ml。在一些實施例中，調配物中之抗體濃度為約70 mg/ml。在一些實施例中，調配物中之抗體濃度為約75 mg/ml。在一些實施例中，調配物中之抗體濃度為約80 mg/ml。在一些實施例中，調配物中之抗體濃度為約85 mg/ml。在一些實施例中，調配物中之抗體濃度為約90 mg/ml。在一些實施例中，調配物中之抗體濃度為約95 mg/ml。在一些實施例中，調配物中之抗體濃度為約100 mg/ml。在一些實施例中，調配物中之抗體濃度為約110 mg/ml。在一些實施例中，調配物中之抗體濃度為約125 mg/ml。 在一些實施例中，向抗體調配物中添加界面活性劑。例示性界面活性劑包括非離子界面活性劑，諸如聚山梨醇酯(例如聚山梨醇酯20、80等)或泊洛沙姆(例如泊洛沙姆188等)。所添加界面活性劑之量使得其減少經調配抗體之聚集及/或使調配物中顆粒之形成減至最少及/或減少吸附。舉例而言，界面活性劑可以約0.001%至約0.5% (w/v)之量存在於調配物中。在一些實施例中，界面活性劑(例如聚山梨醇酯20)為約0.005%至約0.2%、約0.005%至約0.1%、約0.005%至約0.09%、約0.005%至約0.08%、約0.005%至約0.07%、約0.005%至約0.06%、約0.005%至約0.05%、約0.005%至約0.04%、約0.008%至約0.06%、約0.01%至約0.06%、約0.02%至約0.06%、約0.01%至約0.05%，或約0.02%至約0.04%。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.005%或約0.005%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.006%或約0.006%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.007%或約0.007%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.008%或約0.008%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.009%或約0.009%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.01%或約0.01%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.02%或約0.02%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.03%或約0.03%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.04%或約0.04%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.05%或約0.05%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.06%或約0.06%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.07%或約0.07%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.08%或約0.08%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.1%或約0.1%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.2%或約0.2%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.3%或約0.3%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.4%或約0.4%之量存在於調配物中。在某些實施例中，界面活性劑(例如聚山梨醇酯20)以約0.5%或約0.5%之量存在於調配物中。 在一個實施例中，調配物含有上文鑑別之試劑(例如抗體、緩衝液、蔗糖及/或界面活性劑)且基本上不含一或多種防腐劑，諸如苯甲醇、苯酚、間甲酚、氯丁醇及苄索氯銨。在另一實施例中，調配物中可包括防腐劑，尤其調配物為多劑量調配物時。防腐劑之濃度可在約0.1%至約2%，較佳地約0.5%至約1%範圍內。一或多種其他醫藥學上可接受之載劑、賦形劑或穩定劑(諸如Remington's Pharmaceutical Sciences 第16版，Osol, A.編(1980)中所述者)可包括於調配物中，其限制條件為其不會對調配物之所要特徵產生負面影響。可接受之載劑、賦形劑或穩定劑在所使用之劑量及濃度下對接受者無毒且包括：其他緩衝劑；共溶劑；抗氧化劑，包括抗壞血酸及甲硫胺酸；螯合劑，諸如EDTA；金屬錯合物(例如Zn-蛋白質錯合物)；生物可分解聚合物，諸如聚酯；及/或成鹽平衡離子。本文之例示性醫藥學上可接受之載劑進一步包括間質性藥物分散劑，諸如可溶性中性活性玻尿酸酶醣蛋白(sHASEGP)，例如人類可溶性PH-20玻尿酸酶醣蛋白，諸如rHuPH20(HYLENEX® , Baxter International, Inc.)。某些例示性sHASEGP及使用方法(包括rHuPH20)描述於美國專利公開案第2005/0260186號及第2006/0104968號中。在一態樣中，sHASEGP與一或多種額外葡萄糖胺聚糖(諸如硫酸軟骨素酶)組合。 本文之調配物亦可含有一種以上為所治療之特定適應症必需之蛋白質，較佳為具有不會不利地影響其他蛋白質之補充活性的蛋白質。舉例而言，若抗體為抗PDL1，則其可與另一試劑(例如化學治療劑及抗贅生性試劑)組合。 在一些實施例中，評估或量測調配物中之抗體的物理穩定性、化學穩定性或生物活性。此項技術中已知錢描述於中本文實例中之任何方法可用於評估調配物中之抗體的穩定性及生物活性。舉例而言，調配物中之抗體的穩定性可藉由(但不限於)以下量測：尺寸排阻層析(SEC或SE-HPLC)、成像毛細管等電聚焦(ICIEF)、肽定位、小體積遮光(HIAC)分析及毛細管電泳(CE)技術，諸如CE-十二烷基硫酸鈉(CE-SDS)及CE-聚糖分析。在一些實施例中，調配物中之抗體在-20℃下穩定至少約6個月、至少約8個月、至少約10個月、至少約12個月、至少約14個月、至少約16個月、至少約18個月、至少約20個月、至少約21個月、至少約22個月、至少約23個月、至少約24個月、至少約3年或至少約4年。在一些實施例中，調配物中之抗體在2℃至8℃(例如5℃)下穩定至少約6個月、至少約8個月、至少約10個月、至少約12個月、至少約14個月、至少約16個月、至少約18個月、至少約20個月、至少約21個月、至少約22個月、至少約23個月或至少約24個月。在一些實施例中，在儲存後藉由尺寸排阻層析量測調配物中之抗體(亦即抗體單體)之穩定性。在一些實施例中，在儲存後藉由成像毛細管等電聚焦量測調配物中之抗體(亦即抗體單體)之穩定性。在一些實施例中，在-20℃下儲存至少約6個月、至少約12個月、至少約18個月或至少約24個月後，調配物中抗體單體相較於全部蛋白質(例如包括抗體及聚集物)之百分比大於約60%、約65%、約70%、約75%、約80%、約85%、約86%、約87%、約88%、約89%、約90%、約91%、約92%、約93%、約94%或約95%。在一些實施例中，在2℃至8℃(例如5℃)下儲存至少約6個月、至少約12個月、至少約18個月或至少約24個月後，調配物中抗體單體相較於(例如包括抗體及聚集物)之百分比大於約60%、約65%、約70%、約75%、約80%、約85%、約86%、約87%、約88%、約89%、約90%、約91%、約92%、約93%、約94%或約95%。在一些實施例中，在室溫(例如約15℃至25℃)下攪拌至少約2個小時、至少約4個小時、至少約6個小時、至少約8個小時、至少約10個小時、至少約12個小時、至少約14個小時、至少約16個小時、至少約18個小時、至少約20個小時或至少約24個小時後，調配物中抗體單體相較於(例如包括抗體及聚集物)之百分比大於約60%、約65%、約70%、約75%、約80%、約85%、約86%、約87%、約88%、約89%、約90%、約91%、約92%、約93%、約94%或約95%。在一些實施例中，在-20℃下儲存至少約6個月、至少約12個月、至少約18個月或至少約24個月後，調配物中全部聚集物(例如高分子量物質及低分子量物質)之百分比小於約0.1%、約0.2%、約0.3%、約0.4%、約0.5%、約0.6%、約0.7%、約0.8%、約0.9%、約1%、約2%、約3%、約4%、約5%、約6%、約7%、約8%、約9%或約10%中任一者。在一些實施例中，在2℃至8℃(例如5℃)下儲存至少約6個月、至少約12個月、至少約18個月或至少約24個月後，調配物中全部聚集物(例如高分子量物質及低分子量物質)之百分比小於約0.1%、約0.2%、約0.3%、約0.4%、約0.5%、約0.6%、約0.7%、約0.8%、約0.9%、約1%、約2%、約3%、約4%、約5%、約6%、約7%、約8%、約9%或約10%中任一者。在一些實施例中，在室溫(例如約15℃至25℃)下攪拌至少約2個月、至少約4個月、至少約6個月、至少約8個月、至少約10個月、至少約12個月、至少約14個月、至少約16個月、至少約18個月、至少約20個月或至少約24個月後，調配物中全部聚集物(例如高分子量物質及低分子量物質)之百分比小於約0.1%、約0.2%、約0.3%、約0.4%、約0.5%、約0.6%、約0.7%、約0.8%、約0.9%、約1%、約2%、約3%、約4%、約5%、約6%、約7%、約8%、約9%或約10%中任一者。在本文之任何實施例中，穩定調配物可儲存於玻璃瓶、金屬合金容器或靜脈內(IV)袋中。在一些實施例中，金屬合金為316L不鏽鋼或赫史特合金。 用於活體內投與之調配物應無菌。此容易藉由在製備調配物之前或之後經無菌過濾膜過濾而實現。III. 治療及投與抗體調配物之方法 根據已知方法(諸如靜脈內投與(例如以團式或藉由在一段時期內連續輸注)、藉由肌肉內、腹膜內、腦脊髓內、皮下、關節內、滑膜內、鞘內、經口、局部或吸入途徑)向需要用抗體治療之哺乳動物(較佳為人類)投與調配物。在一個實施例中，藉由靜脈內投與向哺乳動物投與調配物。出於此類目的，調配物可例如使用注射器或經IV導管來注射。在一個實施例中，藉由皮下投與向哺乳動物投與調配物。 抗體之適當劑量(「治療有效量」)將視以下而定，例如待治療之病狀、病狀嚴重程度及進程、投與抗體是出於預防目的亦或治療目的、先前療法、患者臨床病史及對抗體之反應、所用抗體之類型及主治醫師之判斷。抗體一次性或經一系列治療適當地投與患者，且可自診斷之日起在任何時間投與患者。可將抗體作為單獨治療投與或聯適合用於治療相關病狀之其他藥物或療法投與。 作為一般建議，藉由一或多次投與向人類投與的治療有效量之抗體將在約0.01至約50 mg/kg患者體重範圍內。在一些實施例中，所用抗體為例如每天投與約0.01至約45 mg/kg、約0.01至約40 mg/kg、約0.01至約35 mg/kg、約0.01至約30 mg/kg、約0.01至約25 mg/kg、約0.01至約20 mg/kg、約0.01至約15 mg/kg、約0.01至約10 mg/kg、約0.01至約5 mg/kg或約0.01至約1 mg/kg。在一些實施例中，抗體以15 mg/kg投與。然而，其他劑量方案亦可適用。在一個實施例中，本文所述之抗PDL1抗體在21天循環的第1天以約100 mg、約200 mg、約300 mg、約400 mg、約500 mg、約600 mg、約700 mg、約800 mg、約900 mg、約1000 mg、約1100 mg、約1200 mg、約1300 mg或約1400 mg之劑量向人類投與。該劑量可以單劑量或多劑量形式(例如2或3個劑量)投與，諸如輸注。組合治療中所投與抗體之劑量相較於單個治療可能降低。此療法之進程容易由習知技術監測。 含有本文所述之抗PDL1抗體的調配物可用於多種活體外及活體內診斷及治療應用中。舉例而言，含有抗體之調配物可向個體投與以治療疾病或病症(例如PD-1及PD-L1相互作用介導之疾病或病症) 在一些實施例中，疾病或病症為癌症。在一些實施例中，癌症為局部晚期癌症或轉移性癌症。在一些實施例中，癌症係選自由以下組成之群：實體腫瘤、血液學癌症、膀胱癌、腦癌、乳癌、結腸癌、結腸直腸癌、胃癌、神經膠質瘤、頭癌、白血病、肝癌、肺癌(例如非小細胞肺癌)、淋巴瘤、骨髓瘤、頸癌、卵巢癌、黑素瘤、胰臟癌、腎癌、唾液癌、胃癌、胸腺上皮細胞癌、甲狀腺癌及頭部及頸部鱗狀細胞癌。在一些實施例中，所治療個體患有PD-L1陽性癌細胞(例如藉由IHC偵測)。 在一些實施例中，疾病或病症為感染。在一些實施例中，感染為持久性感染。在一些實施例中，感染為病毒感染、細菌感染、真菌感染、蠕蟲感染或原蟲感染。在一些實施例中，病毒感染係選自由以下組成之群：巨細胞病毒、埃-巴二氏病毒(Epstein-Barr virus)、B型肝炎、C型肝炎病毒、疱疹病毒、麻疹病毒、流感、人類免疫缺乏病毒、人類嗜T細胞病毒、淋巴球性脈絡叢腦膜炎病毒、呼吸道合胞病毒及/或鼻病毒。在一些實施例中，細菌感染係選自由以下組成之群：螺旋桿菌屬(Helicobacter spp. )、分支桿菌屬(Mycobacterium spp. )、卟啉單胞菌屬(Porphyromonas spp. )、衣原體屬(Chlamydia spp. )、沙門氏菌屬(Salmonella spp. )、李氏菌屬(Listeria spp. )、鏈球菌屬(Streptococcus spp. )、嗜血桿菌屬(Haemophilus spp. )、奈瑟氏菌屬(Neisseria spp. )、克雷伯氏菌屬(Klebsiella spp. )、疏螺旋體屬(Borrelia spp. )、擬桿菌屬(Bacterioides spp. )及密螺旋體屬(Treponema spp. )。在一些實施例中，原蟲感染係選自由以下組成之群：利什曼原蟲屬(Leishmania spp. )、惡性瘧原蟲(Plasmodium falciparum )、住血吸蟲屬(Schistosoma spp. )、弓漿蟲屬(Toxoplasma spp. )、錐蟲屬(Trypanosoma spp. )及絛蟲屬(Taenia spp. )。在一些實施例中，真菌感染係選自由以下組成之群：芽生菌病(blastomycosis)、球孢子菌病(coccidioiodmycosis )、組織胞漿菌病(histoplamsosis )、念珠菌病(candidiasis )、隱球菌病(cryptococcosis )、麯黴病(aspergillossi )、毛黴菌病(mucomycosis )及肺囊蟲病(pneumocystosis )。 在一些實施例中，疾病或病症為發炎疾病。在一些實施例中，發炎疾病係選自由以下組成之群：急性播散性腦脊髓炎、艾迪森氏病(Addison's disease)、阿茲海默氏病(Alzheimer's disease)、僵直性脊椎炎、抗磷脂抗體症候群、動脈粥樣硬化、自體免疫性溶血性貧血、自體免疫肝炎、關節炎、***(Behcet's disease)、伯傑氏病(Berger's disease)、大皰性類天疱瘡、乳糜瀉、查加斯病(Chagas' disease)、膽管炎、克羅恩氏病(Crohn's disease)、皮肌炎、1型糖尿病、絲球體腎炎、古巴士德氏症候群(Goodpasture's syndrome)、移植物抗宿主病、葛瑞夫茲氏病(Graves' disease)、格-巴二氏症候群(Guillain-Barré syndrome)、橋本氏病(Hashimoto's disease)、蕁麻疹、超IgE症候群、特發性血小板減少性紫癜、紅斑狼瘡、狼瘡性腎炎、多發性硬化、重症肌無力、器官移植排斥、帕金森氏病(Parkinson's disease)、天疱瘡、惡性貧血、多發性肌炎、原發性膽汁性肝硬化、牛皮癬、雷諾氏症候群(Raynaud's syndrome)、類風濕性關節炎、硬皮病、休格連氏症候群(Sjögren's syndrome)、顳動脈炎、甲狀腺炎、潰瘍性結腸炎、葡萄膜炎、脈管炎及韋格納肉牙腫病(Wegener's granulomatosis)。 在一些實施例中，含有抗體之調配物可與另一治療劑結合向個體投與治療疾病或病症。舉例而言，為了治療癌症，本文所述之抗PDL1抗體調配物可與另一抗癌治療(例如化學療法或不同抗體治療)結合投與。IV. 製品或套組 在本發明之另一實施例中，提供包含容器之製品或套組，該容器容納本發明之水性醫藥調配物；且視情況提供其使用說明。適合容器包括例如瓶、小瓶、袋及注射器。容器可由多種材料形成，諸如玻璃、塑膠(諸如聚氯乙烯或聚烯烴)或金屬合金(諸如不鏽鋼或赫史特合金)。例示性容器為300 cc金屬合金容器(例如用於在-20℃下儲存)。另一例示性容器可為10-50 cc玻璃瓶(例如用於在2-8℃下儲存)。舉例而言，容器可為10 cc、15 cc、20 cc或50 cc玻璃瓶。容器容納調配物且容器上之標籤或與容器相關之標籤可指示使用說明。該製品可進一步包括其他就商業性及使用者觀點而言合乎需要之材料，包括其他緩衝液、稀釋劑、濾紙、針、注射器及具有使用說明書之包裝插頁。在一些實施例中，製品進一步包含一或多種另一試劑(例如化學治療劑及抗贅生性試劑)。用於一或多種試劑之適合容器包括例如瓶、小瓶、袋及注射器。 認為說明書足以使熟習此項技術者能夠實施本發明。熟習此項技術者將自上文描述知曉除本文中所示及所描述者之外的本發明之各種修改且此等修改在隨附申請專利範圍之範疇內。本文中引用之所有公開案、專利及專利申請案均出於所有目的以引用之方式全部併入本文中。 實例 參考以下實例將更充分瞭解本發明。然而，不應將其視為限制本發明之範疇。應理解本文所述之實例及實施例僅為說明性目的，且熟習此項技術者可對其作出各種修改或改變且其包括於本申請案之精神及範圍及隨附申請專利之範疇內。實例 1 ：抗 PDL1 抗體之調配物研發 抗PDL1抗體(α-PDL1)為源自CHO之去糖基化IgG1抗體，其意欲藉由抑制PDL1/PD1相互作用恢復T細胞功能。研發開始時之攻擊包括CDR區域中或CDR區域附近的潛在Trp氧化及糖基化以及一些甲硫胺酸氧化。穩健性前研究指示pH比先前目標(pH 5.5)高為最佳。目標劑量為固定劑量，但亦涵蓋基於體重之劑量。進行分析研究來分析多種調配物之穩定性且選擇一種調配物(60 mg/mL α-PDL1、20 mM His AcO pH 5.8、120 mM蔗糖、0.04% PS20)。初始調配物研究支持原料藥(DS)及藥品(DS)的高達三年穩定性。方法及材料 製造 α-PDL1 調配物 對經歷超濾/透濾作用之α-PDL1材料進行調配物研發研究。使用10000道爾頓透析卡匣將材料透析至多種調配物緩衝液中。透析後，調整蛋白質濃度達到目標濃度且外加10% PS20儲備溶液以達到目標PS20濃度。將調配材料無菌填充至1 mL填充體積之2 cc Forma Vitrum玻璃瓶中且用13 mm Daikyo 777-1塞密封。樣品在5℃、25℃或40℃下豎直儲存。顏色、外觀及透明度 (CAC) 在室溫下，使用黑色及白色背景，在白色螢光下目測測定樣品顏色、外觀及透明度，如歐洲藥典(EP)法(Council of Europe.European Pharmacopoeia , 2008, 第7版, EP 2.2.2及EP 2.2.1)中所述。用1 mL各測試樣品填充3cc玻璃瓶。使用具有相應樣品體積之陰性對照(純水)進行比較。蛋白質濃度量測 經由使用0.9%生理食鹽水將樣品體積稀釋至約0.5 mg/mL，藉由Agilent 8453分光光度計(Santa Clara, CA.)上之UV吸光度量測測定蛋白質濃度。樣品針對0.9%生理食鹽水預留空白且在約280 nm之Amax 及亦320 nm下量測吸光度。計算Amax 與A320 之間的差異獲得校正Amax ，將其用於使用1.5mL cm-1 mg-1 之吸收性測定最終蛋白質濃度。濁度量測 在Agilent 8453分光光度計上，使用1-cm路徑長度，在石英比色皿中量測樣品在350 nm下之平均光學密度。使用純水作為空白。用於顯微可見粒子之遮光法 (HIAC 分析法 ) 藉由9703型HIAC-Royco (HACH, Loveland, CO.)量測遮光來進行樣品粒子計數。使用PharmSpec v2.0列出各樣品的≥ 2 mm、≥ 5 mm、≥ 10 mm及≥ 25 mm之每毫升平均粒子累積數。每次測試進行總共消耗1.6 mL各樣品的四次讀數，第一讀數捨棄，且其餘3個讀數取平均值。尺寸排阻層析 (SEC 或 SE-HPLC) 藉由使用TosoHaas Bioscience管柱G3000 SWXL (South San Francisco, CA.)之尺寸排阻層析(SEC)在30℃下在Agilent 1200 HPLC (Santa Clara, CA., USA)上測定尺寸變體分佈。所有樣品均未經稀釋以50 μg注射至管柱上且經60分鐘溶離，在280 nm下UV吸收。樣品測試使用兩種不同SEC方法。方法1使用0.20 M磷酸鉀、0.25 M氯化鉀、pH 6.2，而方法2使用0.20 M磷酸鉀、0.25 M氯化鉀，pH 6.2，使用10體積%異丙醇作為移動相。結果以曲線下總面積的峰面積相對百分比形式報導。成像毛細管等電聚焦 (ICIEF) 藉由iCIEF使用具有碳氟化合物塗佈之毛細管濾芯(100 µm × 5 cm)的iCE280分析器(ProteinSimple)評定電荷變體分佈。兩性電解質溶液由0.35%甲基纖維素(MC)、0.75% Pharmalyte 3-10載劑兩性電解質、4.2% Pharmalyte 8-10.5載劑兩性電解質及0.2% pI標記7.40及0.15% pI標記9.77於純水中之混合物組成。陽極電解液為80 mM磷酸，且陰極電解液為100 mM氫氧化鈉，兩者皆含於0.10%甲基纖維素中。樣品稀釋於純水中且以1:100之酶比受質比向各稀釋樣品中添加CpB，隨後在37℃下培育20分鐘。CpB處理樣品與兩性電解質溶液混合，接著藉由引入1500 V電勢持續1分鐘，隨後3000 V電勢持續10分鐘聚焦。藉由使280 nm紫外光通過毛細管且進入電荷耦接裝置數位相機之鏡頭，獲得聚焦α-PDL1電荷變體之影像。接著對此影像進行分析以測定多種電荷變體之分佈。肽定位 使用肽定位技術監測色胺酸(W)及甲硫胺酸(M)氧化。為了產生α-PDL1肽定位，在使蛋白質暴露於二硫蘇糖醇(DTT)及碘乙酸(IAA)後，以還原二硫鍵且改變所得游離硫醇產生羧基甲基衍生物之製程用胰蛋白酶消化蛋白質。所得肽藉由逆相高效液相層析(RP-HPLC)分離且在214 nm下監測。使用ThermoFisher Scientific LTQ-Orbitrap質譜儀藉由經分離消化混合物之LC-MS分析測定胰蛋白酶肽之質量。結果 選擇緩衝液系統 在調配物研發期間，評估兩種緩衝液系統。一種為具有240 mM蔗糖之20 mM組胺酸乙酸鹽，pH 5.5，另一種為200 mM丁二酸精胺酸，pH 5.5。加速穩定性研究揭示α-PDL1在組胺酸乙酸鹽緩衝液中相較於在丁二酸精胺酸緩衝液中具有較佳穩定性(表1)。因此，選擇組胺酸乙酸鹽用於進一步研發調配物。表 1. 30℃下組胺酸乙酸鹽及丁二酸精胺酸緩衝液中ICIEF及SE-HPLC主峰之α-PDL1零級分解速率

Figure 108108366-A0304-0001
註解：所有調配物均在30℃下儲存高達1個月。使用ICIEF及SE-HPLC進行分析；* 150 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、240 mM蔗糖及0.02% (w/v)聚山梨醇酯20，pH 5.5；** 150 mg/mL含α-PDL1之200 mM丁二酸精胺酸、0.02% (w/v)聚山梨醇酯20，pH 5.5。選擇穩定劑 基於蔗糖保護蛋白質免於冷凍/解凍誘發之聚集以及在長期冷凍儲存原料藥(DS)及隨後在2℃至8℃下儲存藥品(DP)期間用作冷凍保護劑的能力，選擇蔗糖(120 mM)作為α-PDL1液體調配物之穩定劑。 在調配物研發期間，20 mM L-組胺酸乙酸鹽，pH 5.5、0.02% (w/v)聚山梨醇酯20及0 mM至120 mM範圍內多種濃度之蔗糖中之50 mg/mL α-PDL1進行五次冷凍/解凍循環。藉由SE-HPLC量測之產物品質指示60 mM蔗糖足以防止冷凍/解凍誘發之α-PDL1 HMWS增加(表2)。此外，120 mM蔗糖顯示維持原料藥在-20℃下冷凍儲存至少6個月時之穩定性(表3)。因此，基於冷凍/解凍研究以及原料藥在-20℃下儲存的長期穩定性之結果，選擇120 mM濃度之蔗糖作為α-PDL1液體調配物之冷凍保護劑。表 2. 蔗糖濃度對α-PDL1 SE-HPLC百分比高分子量物質在冷凍及解凍期間之穩定性的作用
Figure 108108366-A0304-0002
 註解：所有調配物均含有50 mg/mL α-PDL1、20 mM L-組胺酸乙酸鹽、0.02%(w/v)聚山梨醇酯20，pH 5.5。使用以下進行分析：SE-HPLC；F/T = 冷凍/解凍；HMWS = 高分子量物質；SY = 微黃；CL = 清澈；PFVP = 實際上不含可見微粒。表 3. α-PDL1原料藥研發批次之長期穩定性資料
Figure 108108366-A0304-0003
 註解：所有調配物均含有60 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、120 mM蔗糖、0.04% PS20，pH 5.8。此研究使用25cc 316L不鏽鋼小罐；NA=不適用；CAC = 顏色、外觀及透明度；SY=微黃，CL=澄清，PFVP = 實際上不含可見微粒；HMW=高分子量；LMW=低分子量；ICIEF = 成像毛細管等電聚焦；CE-SDS = 十二烷基硫酸鈉毛細管電泳；NT=未測試；TBD = 待測定。預調配穩健性研究：選擇蛋白質濃度、 pH 及聚山梨醇酯 20 濃度 使用部分因子實驗設計(DOE)設計進一步檢查α-PDL1調配物參數對蛋白質穩定性之作用。總共測試12種不同α-PDL1調配物(10種實驗及2種中心點)。研究中變化之三種因素為具有0.5單元間隔的5.0-6.0之pH範圍、40-120 mg/mL的蛋白質濃度範圍及0.005%-0.06%(w/v)的聚山梨醇酯20濃度範圍(表4)。所有調配物均藉由具有120 mM蔗糖之20 mM組胺酸乙酸鹽緩衝，但表4中指示之最後兩種調配物除外。對25 mM組胺酸乙酸鹽調配物進行評估，因為就氧化風險而言其被視為最差案例情況。評估作為備份緩衝液系統的20 mM乙酸鈉緩衝液且與組胺酸乙酸鹽緩衝液比較。調配物儲存於25℃下2個月且40℃下1個月。使用JMP軟體(JMP, 第9版, SAS Institute Inc., Cary, NC)對上文研究之穩定性資料進行調配物參數之間相互作用的統計學分析。表 4. 在DOE研究中評估之α-PDL1原料藥及藥品調配物
Figure 108108366-A0304-0004
 註解：對a 中心點；b 最差案例情況：低蛋白質濃度、高PS20濃度、高組胺酸濃度；c 20 mM乙酸鈉(Na-Ace)緩衝液進行測試。 相較於pH 5.0及5.5，如藉由ICIEF在40℃及25℃下所測定，pH 6.0之調配物主峰損失速率略低(分別圖1A-圖1B及圖2A-圖2B)。ICIEF觀測到濃度對主峰損失無顯著影響。調配物F1之分析顯示在ICIEF中，酸性變體增加主要造成主峰損失，而基本電荷變體造成之峰損失不顯著。在相同儲存條件下，如藉由SE-HPLC在40℃及25℃下所量測，pH 6.0之調配物亦具有較慢單體峰損失速率(分別圖3A-圖3B及圖4A-圖4B)。調配物F1之分析顯示在SEC中，在高溫下(亦即40℃及25℃)，HMWS及LMWS形成皆造成單體損失。SEC及ICIEF pH速率概況皆揭示pH 5.5-6.0為α-PDL1之最佳pH範圍。為了高於pH 5.5之最佳蛋白質穩定性及允許經調配原料藥及藥品中之±0.3 pH單元範圍內，選擇pH 5.8之目標。 上文調配物研究亦揭示如藉由SE-HPLC所測定，在5.0-6.0範圍內之120 mg/mL α-PDL1調配物因為較高HMWS形成而相較於相同pH下的40 mg/mL調配物具有略微較高但不顯著之單體峰損失速率(圖3A-圖3B及圖4A-圖4B)。基於此等資料及為了支持具有改良產物穩定性之調配物及便於患者給藥，選擇60 mg/mL濃度之α-PDL1。 如上文統計分析所指示，觀測到0.005%-0.06%(w/v)濃度範圍之聚山梨醇酯20(PS20)對蛋白質穩定性無影響(圖1-圖4)。 已知聚山梨醇酯20原料中所含之過氧化氫雜質可引起色胺酸(W)及甲硫胺酸(M)氧化。L-組胺酸亦可增加上述氧化風險。藉由肽定位分析含有較高濃度聚山梨醇酯20及L-組胺酸的所選最差案例情況調配物的樣品。分析結果顯示即使較高組胺酸濃度(25mM組胺酸乙酸鹽緩衝液)及較高量PS20(0.06% PS20)之組合亦不展現顯著氧化風險(表5)且組胺酸緩衝液適用於調配α-PDL1。表 5. 藉由肽定位之所選調配物中之Trp百分比及M253 氧化
Figure 108108366-A0304-0005
 為了評定調配物中之PS20在儲存時的可能分解，調配物F1至F10(表4)在40℃下儲存1個月。在25℃下儲存2個月，在5℃下儲存2個月或在5℃下儲存6個月。所評估之調配物在任何升高之溫度(亦即40℃及25℃)及5℃儲存溫度下均未觀測到PS20分解。將所選調配物(亦即F1、F2、F3及F6)之填充體積改為7 ml(高填充)或4 ml(低填充)，接著在5℃下儲存6個月，亦不會對PS20分解速率具有顯著影響(圖5)。 藉由HIAC分析法評定在5℃下儲存6個月之不同調配物中顯微可見粒子(SbVP)之形成，作為穩定性量度(表6)。所測試調配物中未觀測到SbVP中有可量測改變。表 6. 在5℃下儲存6個月後SbVP形成之HIAC資料
Figure 108108366-A0304-0006
 註解：兩個1 mL填充小瓶合併在一起進行小體積HIAC分析。 使用冷凍解凍實驗進一步研究調配物之穩定性。調配物F1至F10(表4)在-20℃下儲存期間進行五次冷凍解凍循環或儲存於5℃之高儲存溫度下0至6個月且隨後藉由SEC及ICIEF分析α-PDL1單體之百分比(圖6A及圖6B)以及調配物中之主峰百分比(圖6C及圖6D)。冷凍解凍循環及在指定時間點儲存後，單體百分比及主峰百分比中未觀測到顯著改變。 藉由在不鏽鋼小罐中在-20℃下儲存高達6個月期間進行五次冷凍解凍循環，隨後藉由CAC、SEC及ICIEF (表7)進行穩定性量測，評定F2調配物中之原料藥穩定性(表4)。在-20℃下儲存6個月後未觀測到改變。表 7. 在-20℃下儲存的不鏽鋼小罐中之原料藥穩定性
Figure 108108366-A0304-0007
 註解：F/T = 冷凍/解凍；SY = 微黃；CL = 澄清。 藉由進行三次冷凍解凍循環，隨後在不鏽鋼小罐或赫史特合金小罐中在-20℃、5℃或25℃下儲存高達3個月，隨後藉由SEC進行穩定性量測，評定含有100 mg/mL α-PDL1、20 mM組胺酸乙酸鹽、120 mM蔗糖、0.04% PS20，pH 5.6之調配物中的原料藥穩定性(圖7A及圖7B)。在pH 5.6下，在不鏽鋼小罐及赫史特合金小罐中儲存未觀測到差異。在-20℃下，在三次冷凍解凍循環後，原料藥穩定高達3個月。儘管不鏽鋼小罐及赫史特合金小罐中存在微小差異，但兩者皆適用於原料藥儲存。 在-5℃、25℃或40℃下，在20cc小瓶中以16 mL填充形式儲存高達3個月，隨後藉由SEC及ICIEF進行穩定性量測，評定含有50 mg/mL α-PDL1、20 mM組胺酸乙酸鹽、120 mM蔗糖、0.04% PS20，pH 5.6之調配物中之藥品穩定性(圖8A及圖8B)。在5℃下儲存三個月後，未觀測到改變。在40℃下，每月的pH 5.6分解速率藉由SEC及ICIEF分析分別為0.66%及22%。 F12調配物中緩衝液之評定指示基於SE-HPLC及ICIEF所量測之主峰分解速率(表8)，乙酸鈉緩衝液提供與組胺酸乙酸鹽緩衝液類似之蛋白質穩定性。兩種測試調配物為50 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、120 mM蔗糖及0.04% (w/v)聚山梨醇酯20，pH 5.5及0 mg/mL含α-PDL1之20 mM乙酸鈉、120 mM蔗糖及0.04% (w/v)聚山梨醇酯20，pH 5.5。表 8. 在40℃下，組胺酸乙酸鹽及乙酸鈉緩衝液中ICIEF及SE-HPLC主峰的α-PDL1零級分解速率
Figure 108108366-A0304-0008
 註解：所有調配物均在40℃下儲存高達1個月。 總體而言，DoE設計穩定性研究揭示在40℃下，ICIEF未觀測到濃度對主峰損失的顯著影響，而較低pH具有略快的主峰速率損失(圖1A-圖1B)。在40℃下，SE-HPLC亦未觀測到顯著相互作用，然而，較高濃度調配物顯示較快單體損失(圖3A-圖3B)。亦發現較低pH具有較快單體速率損失。在25℃下觀測到類似結果(圖2A-圖2B及圖4A-圖4B)。統計分析揭示任何測試調配物參數之間沒有有實際意義的相互作用(聯繫)。攪拌及熱應力研究 對在提高濃度之PS20存在下在玻璃瓶中進行攪拌應力時的藥品穩定性進行研究。在具有0.005%至0.06%範圍內的多種濃度之PS20的2cc玻璃瓶中在1 mL填充中，評定含有20 mM組胺酸乙酸鹽、120 mM蔗糖，pH 5.5中57 mg/mL之調配物。隨後在藉由SEC量測穩定性(圖9A)及量測濁度(圖9B)之前，在70 rpm下，在室溫下攪拌玻璃瓶3天。PS20含量在0.005-0.06%之間的調配物在攪拌期間的穩定性不變。然而，缺乏PS20之調配物顯示由於HMWS增加，單體損失增加。在此實驗中，0.005%的PS20足以保護蛋白質免受玻璃瓶中之攪拌應力影響。 研究藥品調配物(表4)在多種溫度及時間下儲存，接著經受玻璃瓶中之攪拌應力時之穩定性。在2cc玻璃瓶中在1 mL填充中評定各調配物F1-F10。在70 rpm下，在室溫下攪拌玻璃瓶1天，隨後藉由SEC量測穩定性(圖10)。在此實驗中，攪拌對在40℃、25℃或5℃下長時間儲存之藥品的穩定性無影響。 為了支持醫院環境中通常發生的IV袋運輸，使用在具有0.005%- 0.02% (w/v)聚山梨醇酯20的20 mM組胺酸乙酸鹽、240 mM蔗糖，pH 5.5中調配之α-PDL1進行IV袋攪拌研究。藉由注射400-600 mg α-PDL1溶液且使用軌道震盪器在100 rpm下在5℃下攪拌高達6小時，對最常用的250mL含有等滲氯化鈉溶液(0.9% NaCl)之聚氯乙烯(PVC)或聚烯烴(PO) IV袋進行評估。研究結果支持基於重量之給藥且表明在蛋白質溶液中最少需要0.015% (w/v)聚山梨醇酯20以防止在運輸期間形成可見粒子(與蛋白質沈澱有關)(表9)。另外，為了降低聚山梨醇酯20在存放期分解之風險，聚山梨醇酯20濃度自0.02% (w/v)升高至0.04% (w/v)。表 9. 使用不同量的含PS20之α-PDL1藥品的IV袋攪拌研究
Figure 108108366-A0304-0009
 註解：所有調配物50 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、240 mM蔗糖，pH 5.5。使用SE-HPLC進行分析。NT=未測試；CAC = 顏色、外觀及清晰度；CO=無色；CL=澄清；PFVP = 實際上不含可見微粒。α-PDL1 調配物之穩定性評定 對自主細胞庫(Master Cell Bank)及工作細胞庫(Working Cell Bank)產生的在5.2至6.3之pH範圍中在含有20 mM組胺酸乙酸鹽、120 mM蔗糖及0.04% PS20之調配物中之材料進行額外pH篩選(表10)。藉由SE-HPLC及ICIEF分析顯示pH 5.7-6.3在化學上及物理上相當穩定且調配物中pH 5.5-6.3之允許範圍適當(圖11A及圖11B)。較高pH降低單體及主峰分解速率，在約pH 5.7至6.3之間速率變得平坦。表 10. 調配物之pH篩選
Figure 108108366-A0304-0010
 研究調配物賦形劑對α-PDL1調配物中色胺酸(W)及甲硫胺酸(M)氧化之作用。肽定位顯示不存在顯著氧化提高。含有20 mM組胺酸乙酸鹽、120mM蔗糖、0.04% PS20且溶液pH為5.8之調配物顯示當調配物在高溫下儲存一個月時，藥品或原料藥皆無明顯色胺酸及甲硫胺酸氧化提高(表11)。表 11. 藉由肽定位測定的所選調配物中Trp、M253 及M429 氧化之百分比
Figure 108108366-A0304-0011
 註解：所有α-PDL1調配物均含有20 mM L-組胺酸乙酸鹽、120mM蔗糖、0.04% PS20，pH 5.8。 基於此等調配物研究及統計分析之結果，選擇由60 mg/mL含α-PDL1之20 mM組胺酸乙酸鹽、120 mM蔗糖、0.04%聚山梨醇酯20，目標pH 5.8組成之液體調配物進行臨床研究。 將以每位患者1200 mg α-PDL1之均一劑量進行臨床試驗之劑量。選擇20 cc玻璃瓶中標稱20 mL填充(1200mg α-PDL1)之小瓶組態來滿足目標產物概況。 使用含有60 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、120 mM蔗糖及0.02% (w/v)聚山梨醇酯20，pH 5.8的預期調配物進行冷凍/解凍研究。五次冷凍/解凍循環後的分析結果確認120 mM蔗糖保護α-PDL1免於冷凍/解凍誘發之聚集(表12)。預期液體調配物之類似地長期穩定性指示其在2-8℃下穩定超過6個月(表13)。此調配物正在進行連續監測36個月。α-PDL1原料藥原料藥藥品之目標調配物及測試研究範圍顯示於表14中。表 12. α-PDL1原料藥研發批次之代表性冷凍/解凍穩定性資料
Figure 108108366-A0304-0012
 註解：批料PP400L-02142013含有60 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、120 mM蔗糖及0.04% (w/v)聚山梨醇酯20，pH 5.8。CL=澄清；SY=微黃；PFVP = 實際上不含可見微粒；NA = 不適用，ICIEF = 成像毛細管等電聚焦；CE-SDS = 十二烷基硫酸鈉毛細管電泳；HMW=高分子量；LMW=低分子量。表 13. α-PDL1藥物研發批次之穩定性資料
Figure 108108366-A0304-0013
 批料PP400L-02142013-DP含有60 mg/mL含α-PDL1之20 mM L-組胺酸乙酸鹽、120 mM蔗糖及0.04% (w/v) 聚山梨醇酯20，pH 5.8。NA=不適用；CAC = 顏色、外觀及清晰度；SY=微黃，CL=澄清，PFVP = 實際上不含可見微粒；HMW=高分子量；LMW=低分子量；ICIEF = 成像毛細管等電聚焦；CE-SDS = 十二烷基硫酸鈉毛細管電泳，NT=未測試。表 14. α-PDL1原料藥及藥品之目標調配物及測試研究範圍
Figure 108108366-A0304-0014
 因為α-PDL1藥品(60 mg/mL)將在等張氯化鈉溶液(0.9% NaCl)中稀釋後藉由輸注投與，所以在以下模擬製備及投與條件下測試活性成分之相容性及穩定性：1)在2.4 - 9.6 mg/ml範圍中(稀釋後之標稱濃度)在含有0.9% NaCl之輸注袋中稀釋α-PDL1藥品以覆蓋臨床研究中之劑量範圍；2)短期暴露於含有等張氯化鈉溶液之輸注袋(袋產品接觸表面材料由PVC或聚烯烴組成)；3)使用IV輸注管線(產品接觸表面為PVC或聚烯烴)；及4)於在線過濾器中使用0.2 μm(過濾膜為PES)。 在2℃-8℃下儲存24小時或在30℃下暴露於散射光24小時來測試樣品。使用適當穩定性指示方法測試樣品，包括：藉由SE-HPLC及ICIEF測試純度，蛋白質濃度(藉由UV)，藉由不透光度測試顯微可見粒子，顏色、透明度/乳白光及pH (表15)。表 15. 在5℃或30℃下稀釋及儲存24小時，在具有及不具有0.2 μm在線過濾器之0.9% NaCl輸注袋中的α-PDL1穩定性
Figure 108108366-A0304-0015
 表 15 ( 續 ) ： 在5℃或30℃下稀釋及儲存24小時，在具有及不具有0.2 μm在線過濾器之0.9% NaCl輸注袋中的α-PDL1穩定性
Figure 108108366-A0304-0016
 表 15 ( 續 ) ： 在5℃或30℃下稀釋及儲存24小時，在具有及不具有0.2 μm在線過濾器之0.9% NaCl輸注袋中的α-PDL1穩定性
Figure 108108366-A0304-0017
 表 15 ( 續 ) ： 在5℃或30℃下稀釋及儲存24小時，在具有及不具有0.2 μm在線過濾器之0.9% NaCl輸注袋中的α-PDL1穩定性
Figure 108108366-A0304-0018
 表 16. 在5℃下在0.9% NaCl輸注袋中稀釋之α-PDL1的持續高達6小時之攪拌穩定性
Figure 108108366-A0304-0019
 CO=無色，CL=澄清，PFVP = 實際上不含可見微粒，A350 =350 nm處之吸光度 如上文所述的模擬投與研究中之產品測試在測試條件下物理上及化學上穩定。由不同產品接觸材料構成的輸注袋、輸注組、過濾器及/或IV投與助劑在成功取得資格後添加。 除了靜態穩定性之外，使用在具有0.02% PS20之20 mM組胺酸乙酸鹽、120mM蔗糖，pH 5.8中調配之α-PDL1進行IV袋攪拌研究，0.02% PS20潛在地為存放期期間藥品中可觀測到的最低PS20含量。攪拌在2-8℃下使用速度為100 rpm之軌道震盪器進行。資料表明藥品中具有0.02% PS20，α-PDL1在IV袋中稀釋後，在5℃下攪拌後穩定(表16)。實例中所用之抗體序列 α-PDL1 輕鏈可變區
Figure 02_image001
α-PDL1 重鏈可變區
Figure 02_image003
α-PDL1 完全輕鏈
Figure 02_image005
α-PDL1 完全重鏈
Figure 02_image007
Figure 02_image009
I. definition . Before describing the present invention in detail, it should be understood that the present invention is not limited to a specific composition or biological system, which of course may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. As used in the scope of this specification and the accompanying application, unless the content clearly indicates to the contrary, the singular forms "one" and "the" include plural indicators. Thus, for example, reference to "molecule" includes the combination of two or more such molecules as appropriate, and their analogs. As used herein, the term "about" refers to the common error range of individual values that are easily known by those familiar with this technical field. The reference to "about" a value or parameter herein includes (and describes) an embodiment directed to that value or parameter itself. It should be understood that the aspects and embodiments of the present invention described herein include “including” aspects and embodiments, “consisting of aspects and embodiments” and “basically consisting of aspects and embodiments”. The term "pharmaceutical formulation" refers to a formulation in a form that makes the biological activity of the active ingredient effective, and it does not contain additional components that have unacceptable toxicity to the individual to whom the formulation is to be administered. These formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can be reasonably administered to individual mammals to provide an effective dose of all active ingredients. A "sterile" formulation is sterile or free or essentially free of all living microorganisms and their spores. "Frozen" formulations are those with a temperature below 0°C. Generally speaking, frozen formulations are not freeze-dried, nor are they subjected to prior or subsequent freeze-drying. In some embodiments, the frozen formulation contains frozen bulk drug (in a stainless steel tank) or frozen drug (final vial configuration) for easy storage. A "stable" formulation is a formulation in which the protein contained in it substantially maintains its physical and/or chemical stability and/or biological activity after storage. Preferably, the formulation substantially maintains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the expected shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in this technology and, for example, reviewed inPeptide and Protein Drug Delivery , 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A.Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured for a selected period of time at a selected temperature. Stability can be assessed qualitatively and/or quantitatively in many different ways, including assessing aggregate formation (for example, size exclusion chromatography, by measuring turbidity and/or by visual inspection); by using cation exchange chromatography, imaging Capillary isoelectric focusing (icIEF) or capillary zone electrophoresis evaluation; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis comparing shortened antibody and intact antibody; peptide map (such as trypsin or LYS-C) analysis ; Evaluate the biological activity or antigen binding function of antibodies; etc. Instability can involve any one or more of the following: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), splicing/hydrolysis/fragmentation (Such as hinge region fragmentation), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, glycosylation differences, etc. If the protein shows no or very little signs of aggregation, precipitation, and/or denaturation during visual inspection of color and/or transparency or by UV light scattering or by size exclusion chromatography, then the protein in the pharmaceutical formulation "remains Its physical stability". If the chemical stability at a given time allows the protein to be deemed to retain its biological activity as defined below, then the protein in the pharmaceutical formulation "retains its chemical stability." Chemical stability can be assessed by detecting and quantifying chemically altered forms of proteins. The chemical alteration may involve size modification (e.g. splicing), which may be assessed, for example, using size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (for example, caused by desamide), which can be assessed by, for example, ion exchange chromatography or icIEF. If the biological activity of the antibody at a given time is at least about 60% (within analysis error) of the biological activity exhibited during the preparation of the pharmaceutical formulation as determined by the analytical method (eg antigen binding analysis), then the antibody in the pharmaceutical formulation "Retain its biological activity." The other "biological activity" analysis of antibodies is detailed below. As used herein, the "biological activity" of a monoclonal antibody includes the ability of the antibody to bind to an antigen and cause a measurable biological response that can be measured in vitro or in vivo. The "deamidated" monoclonal antibody herein refers to a monoclonal antibody in which one or more asparagine residues have been derived into, for example, aspartic acid or isoaspartic acid. An "oxidized" monoclonal antibody herein refers to a monoclonal antibody in which one or more tryptophan residues and/or one or more methionine have been oxidized. The "glycosylated" monoclonal antibody herein refers to a monoclonal antibody in which one or more lysine residues have been glycosylated. "Easily amylated" antibodies are antibodies that contain one or more residues that have been found to be prone to amylated. "Easily oxidized" antibodies are antibodies that contain one or more residues that have been found to be oxidative. Antibodies that are "easy to aggregate" are antibodies that have been found to aggregate with other antibody molecules, especially after freezing and/or stirring. Antibodies that are "easy to fragment" are antibodies that have been found to be cleaved into two or more fragments, for example, in the hinge region. It is expected to prevent or reduce the amount of deamidation, oxidation, aggregation or fragmentation with respect to monoclonal antibodies formulated in different formulations by "reducing deamidation, oxidation, aggregation or fragmentation". The formulated antibody is preferably substantially pure and desirably substantially homogeneous (e.g., free of contaminating proteins, etc.). "Substantially pure" antibody means that the composition contains at least about 90% by weight, preferably at least about 95% by weight, of the antibody based on the total weight of the protein in the composition. "Substantially homogeneous" antibody means that the composition contains at least about 99% by weight of the antibody based on the total weight of the protein in the composition. "Isotonic" means that the formulation of interest has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using, for example, a vapor pressure or a freezing type osmometer. As used herein, "buffer" refers to a buffer solution that prevents pH from changing by the action of acid-base conjugated components. The buffer of the present invention preferably has about 4.5 to about 7.0, preferably about 5.6 to about 7.0, such as 5.6 to 6.9, 5.7 to 6.8, 5.8 to 6.7, 5.9 to 6.6, 5.9 to 6.5, 6.0, 6.0 to 6.4 or 6.1 To a pH within the range of 6.3. In one embodiment, the buffer has a pH of 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. For example, sodium phosphate is an example of a buffer that will control the pH in this range. As used herein, "surfactant" refers to a surfactant, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (such as polysorbate 20 and polysorbate 80); poloxamer (such as poloxamer 188); Triton; Sodium lauryl sulfate (SDS); Sodium lauryl sulfate; Sodium octyl glycoside; Lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, meat Myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; laurylamino propyl-, cocoamine group Propyl-, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl- or isosteamidopropyl-betaine (e.g. lauramidopropyl) ; Myristylaminopropyl-, palmitoylaminopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurine ; And MONAQUATTM Series (Mona Industries, Inc., Paterson, N.J.); polyethylene glycol, polypropylene glycol and copolymers of ethylene glycol and propylene glycol (such as Pluronics, PF68, etc.); etc. In one embodiment, the surfactant herein is polysorbate 20. From a pharmacological point of view, in the context of the present invention, the "therapeutically effective amount" of an antibody refers to an amount effective to prevent or treat a disorder, wherein the antibody is effective in the treatment of the disorder. "Illness" is any condition that would benefit from treatment with antibodies. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose mammals to the conditions in question. A "preservative" is a compound that may be included in the formulation as appropriate to substantially reduce the effect of bacteria therein, thereby facilitating, for example, the manufacture of a multi-purpose formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexahydroxy quaternary ammonium chloride, benzalkonium chloride (the alkyl group is a long-chain compound A mixture of base ammonium) and benzethonium chloride. Other types of preservatives include aromatic alcohols, such as phenol, butanol, and benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol, resorcinol , Cyclohexanol, 3-pentanol and m-cresol. In one embodiment, the preservative herein is benzyl alcohol. As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of the individual or cell being treated during the course of clinical pathology. The desired therapeutic effects include reducing the rate of disease progression, improving or alleviating disease conditions, and alleviating or improving prognosis. For example, if one or more symptoms related to cancer are reduced or eliminated, including (but not limited to) reducing (or destroying) the proliferation of cancer cells, reducing the symptoms caused by the disease, improving the quality of life of the patient, and reducing the treatment The dosage of other drugs required for the disease, delay the progression of the disease and/or prolong the survival time of the individual will successfully "treat" the individual. As used herein, "delaying disease progression" means delaying, hindering, slowing, retarding, stabilizing, and/or delaying the development of a disease (such as cancer). Depending on the history of the disease and/or the individual being treated, the length of this delay can vary. As is obvious to those familiar with the technology, a sufficient or significant delay can actually cover prevention, where the individual does not develop the disease. For example, advanced cancers (such as the development of metastases) can be delayed. An "effective amount" is at least the minimum amount required to achieve a measurable improvement or prevention of a specific disease. The effective amount herein may vary according to factors such as the patient's disease condition, age, sex, and weight, and the ability of the antibody to cause a desired response in the individual. An effective amount is also an amount where the beneficial effects of the treatment exceed any toxic or harmful effects of the treatment. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, which includes the biochemical, tissue, and/or behavioral symptoms of the disease, and other manifestations during the development of the disease. Complications and intermediate pathological phenotypes. For therapeutic use, beneficial or desired results include such as reducing one or more symptoms caused by the disease, improving the quality of life of the person suffering from the disease, reducing the dose of other drugs needed to treat the disease, enhancing the effect of another drug (such as via a target To), delay disease progression and/or prolong survival time. In the case of cancer or tumor, an effective amount of the drug can have the following effects: reduce the number of cancer cells; reduce the size of the tumor; inhibit (that is, slow down or ideally stop) the infiltration of cancer cells into the surrounding organs; inhibit (That is, to slow down or ideally stop) tumor metastasis; inhibit tumor growth to a certain extent; and/or alleviate one or more symptoms related to the disease to a certain extent. The effective amount can be administered in one or more administrations. For the present invention, the effective amount of the drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical context, the effective amount of a drug, compound, or pharmaceutical composition can be achieved with or without combination with another drug, compound, or pharmaceutical composition. Therefore, in the case of administering one or more therapeutic agents, an "effective dose" can be considered, and if combined with one or more other agents, the desired result can be achieved or achieved, then a single agent can be regarded as being given in an effective amount. As used herein, "combined" refers to the administration of one mode of treatment in addition to another mode of treatment. Therefore, "combination" refers to the execution of one treatment mode before, during or after another treatment mode is performed on the individual. A "disorder" is any condition that will benefit from treatment, including but not limited to chronic and acute conditions or diseases, including those pathological conditions that predispose mammals to the condition of interest. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders related to a certain degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor. As used herein, "tumor" refers to all neoplastic cell growth and proliferation regardless of malignant or benign, and all precancerous and cancerous cells and tissues. As referred to herein, the terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive. The terms "cancer" and "cancerous" refer to or describe the physiological condition of mammals that is usually characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of this type of cancer include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, colon cancer, rectum Cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial disseminated melanoma, malignant Small mole melanoma, acral freckle melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (including mild/follicular non-Hodgkin's lymphoma (non-Hodgkin's lymphoma, NHL), small lymphocytic (SL) NHL, moderate/follicular NHL, moderate diffuse NHL, severe immunoblast NHL, severe lymphoblast NHL, severe small non-cleaved cell NHL, giant tumor NHL, Mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's Macroglobulinemia (Waldenstrom's Macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic Myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with blastocytosis, edema (such as edema associated with brain tumors), Meigs' syndrome, brain Cancer and head and neck cancer and related metastases. In certain embodiments, cancers suitable for treatment with the antibody of the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin’s lymphoma (NHL), renal cell Carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid, head and neck cancer, ovarian cancer, mesothelioma and multiple myeloma. In some embodiments, the cancer line is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In some embodiments, the cancer line is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast cancer, including metastatic forms of these cancers. "Chemotherapeutic agents" are compounds suitable for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN® ); Sulfonic acid alkyl esters, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone ), metedopa and uredopa; ethylene imine and methyl melamine, including altretamine, trimethylene melamine, trimethylene phosphamide, trimethylene Thiophosphamide and trimethylolmelamine; polyacetamide (especially bullatacin and bullatacinone); δ-9-tetrahydrocannabinol (dronnabinol ( dronabinol), MARINOL® ); β-lapachone (beta-lapachone); lapachol (lapachol); colchicine; betulinic acid; camptothecin (including the synthetic analog topotecan (HYCAMTIN)® ), CPT-11 (irinotecan, CAMPTOSAR® ), acetylcamptothecin, scopolamine and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including adozelesin), Carzelesin and bizelesin synthetic analogs); Podophyllotoxin; Podophyllic acid; Teniposide; Nostoc cyclic peptide (especially Nostoc cyclin 1 and Nostoc Cyclic peptide 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; Sarcodictyin (sarcodictyin); spongistatin (spongistatin); nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, Ifosfamide (ifosfamide), chlorethamine (mechlorethamine), chlorethamine oxide hydrochloride, melphalan (melphalan), new nitrogen mustard (novembichin), phenesterine (phenesterine), prednisolone Mustard (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil mustard); nitrosoureas, such as carmustine (carmustine), chlorozotocin (chlorozotocin), formustine (fotemustine) , Lomustine (lomustine), nimustine (nimustine) and ramustine (ranimnustine); antibiotics, such as enediyne antibiotics (such as calicheamicin (calicheamicin), especially calicheamicin γ1I and calico Spectinomycin ωIl (see, for example, Nicolaou et al.,Angew. Chem Intl. Ed. Engl. , 33: 183-186 (1994)); CDP323, an oral α-4 integrin inhibitor; dynemicin (dynemicin), including danomycin A; esperamicin; and new Tumor suppressor protein chromophore and related chromoprotein enediyne antibiotic chromophore); aclacinomysin; actinomycin; authramycin; azaserine; Bleomycin (bleomycin); Actinomycin C (cactinomycin); Carabicin (carabicin); Caminomycin (caminomycin); Carzinophilin (carzinophilin); Chromomycin (chromomycin); Actinomycin d ( dactinomycin); daunorubicin; (detorubicin); 6-diazo-5-oxo-L-ortho-leucine; cranberry (doxorubicin) (including ADRIAMYCIN®, (N-morpholine) Base)-Cranberry, Cyano-(N-morpholinyl)-Cranberry, 2-Pyrololinyl-Cranberry, Cranberry HCl Liposome Injection (DOXIL®), Lipid Cranberry TLC D-99 (MYOCET®), pegylated lipid cranberries (CAELYX®) and deoxygenated cranberries); epirubicin (epirubicin); esorubicin (esorubicin); idarubicin; Marcellomycin (marcellomycin); mitomycin (mitomycin), such as mitomycin C; mycophenolic acid (mycophenolic acid); nogalamycin (nogalamycin); olivinemycin (olivomycin); Peiro mold Peplomycin; Porfiromycin; Puromycin; Quelamycin; Rodorubicin; Streptonigrin; Streptozotocin (streptozocin); tubercidin; ubenimex; zinostatin; zorubicin; antimetabolites, such as methotrexate, gemcitabine (gemcitabine) (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone and 5-fluorouracil (5-FU); Cobb Statin (comb retastatin); folate analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6 Mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azuridine, carmofur, Cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluridine (doxifluridine), enocitabine (enocitabine), fluorouridine (floxuridine); Androgen (androgen), such as carudosterone ( calusterone), dromostanolone propionate (epitiostanol), mepitiostane, testolactone; anti-adrenal drugs, such as aminoglutethimide, Mito Mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminacetin (aminolevulinic acid); eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; Colchicine (demecolcine); diacrquinone (diaziquone); elformithine (elformithine); elliptinium acetate (elliptinium acetate); epothilone (epothilone); etoglucid; gallium nitrate ); hydroxyurea; lentinan; lonidainine; maytansinoid, such as maytansine and ansamitocin; mitoguazone ; Mitoxantrone (mitoxantrone); Mopidanmol (mopidanmol); Nitraerine (nitraerine); Pentostatin (pe ntostatin); phenamet; pirarubicin; losoxantrone; 2-ethylhydrazine; procarbazine; PSK® polysaccharide complex (JHS Natural Products , Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triimine quinone ( triaziquone); 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A), roridin A and serpentine Anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; two Mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, such as Pacific Paclitaxel (TAXOL)® , Bristol-Myers Squibb Oncology, Princeton, N.J.), the albumin engineered nanoparticle formulation of paclitaxel (ABRAXANETM ) And docetaxel (TAXOTERE® , Rhome-Poulene Rorer, Antony, France); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum reagents such as cisplatin, oxaliplatin ( Such as ELOXATIN® and carboplatin; Vincas, which prevents the polymerization of tubulin to form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), Vindesine (ELDISINE®, FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone ; Leucovorin (leucovorin); mitoxantrone (novantrone); edatrexate (edatrexate); daunorubicin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate) ; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid, including bexarotene (TARGRETIN®); Bisphosphine Acid salts, such as clodronate (e.g. BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate ( zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronic acid Salt (risedronate) (ACTONEL®); troxacitabine (1,3-dioxolanucleoside cytosine analogue); antisense oligonucleotides, especially to inhibit signal transduction involved in abnormal cell proliferation The gene expression in the pathway, such as PKC-α, Raf, H-Ras and epidermal growth factor receptor (EGF-R) (for example, erlotinib (Tarceva)TM )); and VEGF-A to reduce cell proliferation; vaccines, such as THERATOPE® vaccine and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; type 1 topoisomerase inhibitor (such as LURTOTECAN®); rmRH (for example, ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib; SUTENT®, Pfizer); perifosine; COX-2 inhibition (E.g. celecoxib or etoricoxib); proteasome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib ) (R11577); Sorafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitor; Tyrosine Kinase inhibitors; serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE®); farnesyl transferase inhibitors, such as Lonafani ( lonafarnib) (SCH 6636, SARASARTM ); and a pharmaceutically acceptable salt, acid or derivative of any of the above; and a combination of two or more of the above, such as CHOP, cyclophosphamide, cranberry, vincristine and polynylon The abbreviation of combination therapy; and FOLFOX, using oxaliplatin (ELOXATINTM ) An abbreviation for a treatment regimen in combination with 5-FU and methytetrahydrofolate, and a pharmaceutically acceptable salt, acid or derivative of any of the above; and a combination of two or more of the above. As defined herein, chemotherapeutic agents include "antihormonal agents" or "endocrine therapeutic agents" used to modulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth. It can be the hormone itself, including but not limited to: anti-estrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including NOLVADEX® tamoxifen), ranoxifen Phenol (raloxifene), troloxifene (droloxifene), 4-hydroxy tamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY117018, onapristone and FARESTON.cndot . Toremifene; an aromatase inhibitor that inhibits the enzyme aromatase, which regulates the production of estrogen in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® methyl acetate Progesterone, AROMASIN® exemestane, Formestanie, Fadrozole, RIVISOR® Vorozole, FEMARA® Letrozole and ARIMIDEX® Anastrozole ( anastrozole); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxa Troxacitabine (1,3-dioxolan nucleoside cytosine analogue); antisense oligonucleotides, especially those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α , Raf and H-Ras; ribonuclease, such as VEGF expression inhibitor (such as ANGIOZYME® ribonuclease) and HER2 expression inhibitor; vaccine, such as gene therapy vaccine, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® Type 1 topoisomerase inhibitor; ABARELIX® rmRH; Vinorelbine and Esperamicin (see U.S. Patent No. 4,675,187), and any of the above Pharmaceutically acceptable salts, acids or derivatives of the above; and a combination of two or more of the above. "Growth inhibitory agent" when used herein refers to a compound or composition that inhibits cell growth in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces the proliferation of cells expressing the antigen to which the antibody binds. In another embodiment, the growth inhibitor can be a growth inhibitor that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include agents that block cell cycle progression (other than S phase), such as agents that induce G1 arrest and M phase arrest. Classic M-phase blockers include vinblastines (vincristine and vinblastine), taxanes and type II topoisomerase inhibitors (such as cranberries, epirubicin, daunorubicin, Etoposide and Bleomycin). The agents that cause G1 arrest also penetrate into S phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, methotrexate, cisplatin, methotrexate, 5-Fluorouridine and ara-C. Other information can be found in Mendelsohn and Israel eds, The Molecular Basis of Cancer, Chapter 1, titled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al. (W.B. Saunders, Philadelphia, 1995), for example, page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel derived from European yew (TAXOTERE® , Rhone-Poulenc Rorer) is paclitaxel (TAXOL® , Bristol-Myers Squibb) semi-synthetic analogue. Paclitaxel and docetaxel promote the assembly of self-tubulin dimers into microtubules and stabilize the microtubules by preventing depolymerization, which will result in the inhibition of mitosis in cells. "Radiation therapy" means the use of directed gamma or beta rays to induce sufficient damage to cells in order to limit the ability of the cells to function normally or completely destroy the cells. It should be understood that a variety of methods for determining the therapeutic dose and duration are known in the art. The typical treatment is given in a single administration and the typical dosage is in the range of 10 to 200 units (Gray) per day. For treatment, "individual" refers to any animal classified as a mammal, including humans, domestic animals, and farm animals, as well as zoo animals, sports animals, or pet animals, such as dogs, horses, cats, cows, etc. The mammal is preferably a human. The term "antibody" herein is used in the broadest sense and especially encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiple antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity . "Isolated" antibodies are antibodies that have been identified and separated from components of their natural environment and/or recovered from them. The pollutant components of the natural environment are substances that interfere with antibody research, diagnostic or therapeutic use, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody as determined by, for example, the Lowry method, and in some embodiments to greater than 99% by weight; (2) to be sufficient Obtain the degree of at least 15 N-terminal residues or internal amino acid sequences determined by using, for example, a spinning cup sequenator; or (3) to by using, for example, Coomassie blue (Coomassie blue). Blue) or silver staining is determined by SDS-PAGE under reducing or non-reducing conditions to determine the degree of homogeneity. Since at least one component of the antibody's natural environment will not be present, the isolated antibody includes the antibody in situ in recombinant cells. However, isolated antibodies will usually be prepared by at least one purification step. A "natural antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds in the heavy chain of different immunoglobulin isotypes is different. Each heavy chain and light chain also have regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH ), followed by a large number of constant domains. Each light chain has a variable domain (VL ) And has a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains. The term "constant domain" refers to a part of an immunoglobulin molecule that has an amino acid sequence that is more conservative than other parts of an immunoglobulin (variable domain containing an antigen-binding site). Constant domain contains heavy chain CH 1. CH 2 and CH 3 domains (collectively referred to as CH) and the CHL (or CL) domain of the light chain. The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain can be called "VH ". The variable domain of the light chain can be called "VL ". These domains are usually the most variable parts of antibodies and contain antigen binding sites. The term "variable" refers to the fact that the sequence of certain parts of the variable domain varies widely between antibodies and is used for the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three sections called hypervariable regions (HVR) in the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of variable domains are called the framework regions (FR). The variable domains of the primary heavy chain and the light chain each contain four FR regions that mainly adopt the β-sheet configuration. These FR regions are connected by three HVRs, which form a loop connecting the β-sheet structure, and in some cases It forms part of the β-sheet structure. The HVR in each chain is tightly combined with the HVR in the other chain by the FR region to form the antigen binding site of the antibody (see Kabat et al.,Sequences of Proteins of Immunological Interest , 5th edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, such as involving antibodies in antibody-dependent cytotoxicity. The "light chain" of an antibody (immunoglobulin) from any mammalian species can be assigned to one of two distinct types called kappa and lambda based on the amino acid sequence of its constant domain. The term "isotype" or "subclass" of IgG as used herein means any subclass of immunoglobulin defined by the chemical and antigenic characteristics of the constant region. Antibodies (immunoglobulins) can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five main classes of immunoglobulin: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1 , IgG2 , IgG3 , IgG4 , IgA1 And IgA2 . The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described in, for example, Abbas et al.Cellular and Mol. Immunology , 4th edition. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by the covalent or non-covalent association of the antibody with one or more other proteins or peptides. The terms "full-length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to indicate a substantially complete form of the antibody rather than an antibody fragment as defined below. These terms especially refer to antibodies with heavy chains containing an Fc region. For the present invention, "naked antibodies" are antibodies that do not bind to cytotoxic moieties or radioactive labels. "Antibody fragments" include parts of intact antibodies, preferably their antigen binding regions. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 And Fv fragments; bifunctional antibodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site; and an "Fc" fragment, the name reflects its ability to be easily crystallized. Pepsin treatment produces F(ab') with two antigen combination sites and still capable of cross-linking with antigen2 Fragment. "Fv" is the smallest antibody fragment that contains a complete antigen binding site. In one embodiment, the double-chain Fv substance is composed of a dimer of a heavy chain variable domain and a light chain variable domain in tight non-covalent association. In single-chain Fv (scFv) substances, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and heavy chain can be similar to the double-chain Fv substance The "dimeric" structure is associated. In this configuration, the three HVRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. In total, six HVRs confer antigen binding specificity to antibodies. However, even a single variable domain (or half of the Fv containing only three HVRs specific to the antigen) still has the ability to recognize and bind the antigen, but the affinity is lower than the entire binding site. The Fab fragment contains the variable domain of the heavy chain and the variable domain of the light chain and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by adding a small number of residues (including one or more cysteines from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain. Fab'-SH refers to Fab' in which the cysteine residue of the constant domain has a free thiol group. F(ab')2 Antibody fragments were originally manufactured as a pair of Fab' fragments with cysteine in the hinge region in the middle. Other chemical couplings of antibody fragments are also known. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally speaking, the scFv polypeptide additionally includes a polypeptide linker between the VH and VL domains that enables the scFv to form a structure required for antigen binding. For scFv reviews, see, for example, Pluckthün,The Pharmacology of Monoclonal Antibodies , Volume 113, Rosenburg and Moore eds, (Springer-Verlag, New York, 1994), pages 269-315. The term "bifunctional antibody" refers to antibody fragments with two antigen binding sites, these fragments comprising a heavy chain variable domain (VL) connected to the same polypeptide chain (VH-VL) VH). By using linkers that are too short to allow pairing between two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 1993/01161; Hudson et al.Nat. Med. 9:129-134 (2003); and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al.,Nat. Med. 9:129-134 (2003). The term "monoclonal antibody" as used herein refers to antibodies obtained from a group of substantially homologous antibodies, for example, the individual antibodies constituting the population are identical except for possible mutations (such as naturally occurring mutations) that may exist in small amounts. Therefore, the modifier "monoclonal" refers to the characteristics of the antibody when it is not a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies that include a target-binding polypeptide sequence, wherein the target-binding polypeptide sequence is obtained by a method that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method may be to select a unique clone from a plurality of clones (such as a pool of fusion tumor clones, phage clones, or recombinant DNA clones). It should be understood that the selected target binding sequence can be further changed, for example, to improve the affinity to the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, and produce multispecific antibodies. Etc., and an antibody containing an altered target binding sequence is also a monoclonal antibody of the present invention. In contrast to multi-strain antibody preparations which usually include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody system of the monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibody preparations are advantageous because they are usually not contaminated by other immunoglobulins. The modifier "monolone" indicates the characteristics of the antibody system obtained from a substantially homogeneous antibody population, and should not be understood as requiring the production of antibodies by any specific method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, for example, the fusion tumor method (such as Kohler and Milstein,Nature , 256:495-97 (1975); Hongo et al.,Hybridoma , 14 (3): 253-260 (1995); Harlow et al.,Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd edition. 1988); Hammerling et al.,Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)); recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567); phage display technology (see, for example, Clackson et al.,Nature , 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Sidhu et al.,J. Mol. Biol. 338(2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004); and technology for producing human or human-like antibodies with part or all of human immunoglobulin loci or genes encoding human immunoglobulin sequences in animals (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al.,Year in Immunol. 7:33 (1993); US Patent No. 5,545,807; No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425 and No. 5,661,016; Marks et al.,Bio/Technology 10: 779-783 (1992); Lonberg et al.,Nature 368: 856-859 (1994); Morrison,Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger,Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13: 65-93 (1995). Monoclonal antibodies herein especially include "chimeric" antibodies, in which a part of the heavy chain and/or light chain is identical to or homologous to the corresponding sequence in an antibody obtained from a specific species or belonging to a specific antibody class or subclass, and The remainder of the chain is identical to or homologous to the corresponding sequences in antibodies obtained from another species or belonging to another antibody class or subclass and fragments of such antibodies, as long as they exhibit the required biological activity (see, for example, the United States Patent No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® Antibodies, where the antigen binding region of the antibody is obtained from an antibody produced by, for example, immunizing rhesus monkeys with the antigen of interest. The "humanized" form of non-human (e.g., murine) antibodies is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulin. In one embodiment, the humanized antibody is a residue of HVR from the recipient replaced with HVR from a non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate) and has Human immunoglobulin (recipient antibody) of residues with the desired specificity, affinity and/or ability. In some cases, FR residues of human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues not seen in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FR is the FR of the human immunoglobulin sequence. The humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. For other details, see, for example, Jones et al.,Nature 321:522-525 (1986); Riechmann et al.,Nature 332:323-329 (1988); and Presta,Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton,Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409. "Human antibodies" are antibodies that have amino acid sequences that correspond to those of antibodies produced by humans and/or have been prepared using any of the techniques for preparing human antibodies as disclosed herein. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Various techniques known in the art (including phage display libraries) can be used to produce human antibodies. Hoogenboom and Winter,J. Mol. Biol ., 227:381 (1991); Marks et al.,J. Mol. Biol ., 222:581 (1991). Cole et al.,Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985); Boerner et al.,J. Immunol. , 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel,Curr. Opin. Pharmacol. , 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., immunized xenomice) that has been modified to produce such antibodies in response to an antigen challenge but whose endogenous loci are no longer available ( About XENOMOUSETM See, for example, US Patent Nos. 6,075,181 and 6,150,584). For human antibodies produced by human B-cell fusion tumor technology, see, for example, Li et al.Proc. Natl. Acad. Sci. USA , 103:3557-3562 (2006). A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than a homologue of the antigen from a second mammalian species. Generally, species-dependent antibodies "specifically bind" to human antigens (for example, the binding affinity (Kd) value does not exceed about 1×10-7 M, preferably not more than about 1×10-8 M and preferably no more than about 1×10-9 M), but the binding affinity for the homologue of the antigen from the second non-human mammalian species is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than its binding affinity for the human antigen. The species-dependent antibody may be any of the various types of antibodies as defined above, but is preferably a humanized or human antibody. As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region in an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop. Generally speaking, an antibody contains six HVRs; three are located in the VH (H1, H2, H3), and three are located in the VL (L1, L2, L3). Among the native antibodies, H3 and L3 show the most diversity among the six HVRs, and it is believed that H3, in particular, plays a unique role in conferring fine specificity on antibodies. See, for example, Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu,Methods in Molecular Biology 248:1-25 (Lo, Human Press, Totowa, NJ, 2003). In fact, a naturally occurring camel antibody consisting only of heavy chains is functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al.,Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996). Many HVR narratives are used and covered in this article. The Kabat complementarity determining region (CDR) is based on sequence variability and is most commonly used (Kabat et al.,Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia also pointed out the location of structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on the analysis of available complex crystal structures. The residue annotations for each of these HVRs are as follows. Ring Kabat AbM Chothia contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat number) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia code) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101 HVR can include the following "extended HVR": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26 in VH -35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3). The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions. "Framework" or "FR" residues are those variable domain residues other than HVR residues as defined herein. The terms "number of variable domain residues as in Kabat" or "number of amino acid positions as in Kabat" and their variants refer to the heavy chain variable domain or variable domain of Kabat et al. (supra) used to compile antibodies. The numbering system for light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which is equivalent to simplifying or inserting the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to Kabat residue 52a) and an inserted residue after heavy chain FR residue 82 (e.g. according to Kabat Residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by comparing the antibody sequence with the homology region of the "standard" Kabat numbering sequence. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al.,Sequences of Immunological Interest . 5th edition. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (for example, the EU index reported in Kabat et al. (supra)). "EU index in Kabat" refers to the residue number of human IgG1 EU antibody. The expression "linear antibody" refers to Zapata et al. (1995Protein Eng , 8(10):1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. As used herein, the term "specifically binds" or "specifically" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, in the presence of a heterogeneous population of molecules including biomolecules Determine the existence of the goal. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds to this target with greater affinity (affinity, avidity), easier and/or longer duration than other targets. In one embodiment, as measured by radioimmunoassay (RIA), the degree of binding of the antibody to the unrelated target is less than about 10% of the binding of the antibody to the target. In some embodiments, the dissociation constant (Kd) of the antibody that specifically binds to the target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, antibodies specifically bind to epitopes on proteins that are conserved among proteins from different species. In another embodiment, specific binding may include (but not necessarily) exclusive binding.II. Antibody formulations and preparations The invention here is about stable aqueous formulations comprising antibodies, such as anti-PDL1 antibodies. In some embodiments, the formulation includes an antibody (such as a monoclonal antibody), sucrose, a buffer, and a surfactant, wherein the pH of the formulation is about 5.0 to about 7.0. In some embodiments, the antibody in the formulation (such as the anti-PDL1 antibody described herein) is in an amount of about 40 mg/ml to about 125 mg/ml. In some embodiments, the buffer is histidine (e.g., histidine acetate) or sodium acetate. In some embodiments, the buffer in the formulation is at a concentration of about 15 mM to about 25 mM. In some embodiments, the sucrose in the formulation is about 60 mM to about 240 mM. In some embodiments, the surfactant in the formulation is polysorbate (e.g., polysorbate 20). In some embodiments, the polysorbate in the formulation is at a concentration of about 0.005% (w/v) to about 0.06% (w/v). In some embodiments, the pH of the formulation is about 5.0 to about 6.3. In some embodiments, provided herein is a stable aqueous pharmaceutical formulation comprising an anti-PDL1 monoclonal antibody at a concentration of about 40 mg/ml to about 125 mg/ml, and histamine at a concentration of about 15 mM to about 25 mM Acid acetate or sodium acetate, sucrose with a concentration of about 60 mM to about 240 mM, polysorbate with a concentration of about 0.005% (w/v) to about 0.06% (w/v), and a pH of about 5.0 to Approximately 6.3. In some embodiments, the formulation includes an anti-PDL1 monoclonal antibody in an amount of about 125 mg/ml, sucrose at a concentration of about 240 mM, and a pH of about 5.5. In some embodiments, the formulation comprises an anti-PDL1 monoclonal antibody in an amount of about 60 mg/ml, sucrose at a concentration of about 120 mM, and a pH of about 5.8. In some embodiments, the antibody in the formulation is stable at -20°C for at least about 6 months, at least about 12 months, at least about 18 months, at least two years, at least three years, or at least four years. In some embodiments, the antibody in the formulation is stable at 2-8°C for at least about 6 months, at least about 12 months, at least about 18 months, at least two years, or at least three years. In some embodiments, after storage, the antibody retains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. % Biological activity (such as binding to the target or therapeutic efficacy) before storage (that is, when the pharmaceutical formulation is prepared). In certain embodiments, the formulation is stable at about 40°C for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, 28 days, or more than 28 days . In certain embodiments, the formulation is stable at about 40°C for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more than 8 weeks. In certain embodiments, the formulation is stable at about 25°C for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months Month, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more than 24 months. In certain embodiments, the formulation is stable at about 5°C for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months Month, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more than 24 months. In certain embodiments, the formulation is stable at about -20°C for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months Months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months , 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 Months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months , 47 months, 48 months or more than 48 months. In certain embodiments, the formulation is stable at 5°C or -20°C for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months , 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 Months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months , 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 Months, 47 months, 48 months or more than 48 months. In addition, the formulation is preferably stable after freezing (to, for example, -20°C, -40°C, or -70°C) and thawing the formulation, such as after 1, 2, 3, 4, or 5 freezing and thawing cycles.A. Antibody ( Such as anti PDL1 Antibody ) In some embodiments, the antibody in the formulation contains at least one tryptophan (e.g., at least two, at least three, or at least four) in the heavy chain and/or light chain sequence. In some embodiments, the amino acid tryptophan is in the CDR region, framework region and/or constant region of the antibody. In some embodiments, the antibody contains two or three tryptophan residues in the CDR regions. In some embodiments, the antibody in the formulation is an anti-PDL1 antibody. PD-L1 (progressive cell death 1 ligand 1) is also known as PDL1, B7-H1, B7-4, CD274 and B7-H. It is a transmembrane protein, and its interaction with PD-1 inhibits T Cell activation and cytokine production. In some embodiments, the anti-PDL1 antibodies described herein bind to human PD-L1. Examples of anti-PDL1 antibodies that can be formulated using the formulations described herein are described in PCT patent applications WO 2010/077634 A1 and US 8,217,149, which are incorporated herein by reference. In some embodiments, the anti-PDL1 antibody can inhibit the binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PDL1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab')2 Fragment. In some embodiments, the anti-PDL1 antibody is a humanized antibody. In some embodiments, the anti-PDL1 antibody is a human antibody. The anti-PDL1 antibodies described in WO 2010/077634 A1 and US 8,217,149 can be formulated in the formulations described herein. In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 30, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 31. In one embodiment, the anti-PD-L1 antibody contains a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2 and HVR-H3 sequences, wherein: (a) HVR-H1 sequence is GFTFSX1 SWIH (SEQ ID NO: 11); (b) HVR-H2 sequence is AWIX2 PYGGSX3 YYADSVKG (SEQ ID NO: 12); (c) The sequence of HVR-H3 is RHWPGGFDY (SEQ ID NO: 13); In addition: X1 Is D or G; X2 Is S or L; X3 For T or S. In a particular aspect, X1 Is D; X2 S and X3 Is T. In another aspect, the polypeptide is further included according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)- (HC-FR4) The juxtaposed variable region heavy chain framework sequence between HVRs. In another aspect, the framework sequence is derived from the human common framework sequence. In another aspect, the framework sequence is the VH subgroup III common framework. In another aspect, at least one framework sequence is as follows: HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 14) HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 15) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 17). In another aspect, the heavy chain polypeptide is further combined with the variable region light chain comprising HVR-L1, HVR-L2 and HVR-L3, wherein: (a) HVR-L1 sequence is RASQX4 X5 X6 TX7 X8 A (SEQ ID NO: 18); (b) HVR-L2 sequence is SASX9 LX10 S (SEQ ID NO: 19); (c) HVR-L3 sequence is QQX11 X12 X13 X14 PX15 T (SEQ ID NO: 20); In addition: X4 Is D or V; X5 Is V or I; X6 Is S or N; X7 Is A or F; X8 Is V or L; X9 Is F or T; X10 Is Y or A; X11 Is Y, G, F or S; X12 Is L, Y, F or W; X13 Y, N, A, T, G, F or I; X14 Is H, V, P, T or I; X15 It is A, W, R, P or T. In another aspect, X4 Is D; X5 Is V; X6 Is S; X7 Is A; X8 Is V; X9 Is F; X10 Is Y; X11 Is Y; X12 Is L; X13 Is Y; X14 Is H; X15 Is A. In another aspect, the light chain is further included according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3) -(LC-FR4) the juxtaposed variable region light chain framework sequence between HVRs. In another aspect, the framework sequence is derived from a human common framework sequence. In another aspect, the framework sequence is a VL κ I common framework. In another aspect, at least one framework sequence is as follows: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:23) LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 24). In another embodiment, an isolated anti-PDL1 antibody or antigen-binding fragment is provided, which comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain includes HVR-H1, HVR-H2 and HVR-H3, among which: (i) HVR-H1 sequence is GFTFSX1 SWIH; (SEQ ID NO: 11) (ii) HVR-H2 sequence is AWIX2 PYGGSX3 YYADSVKG (SEQ ID NO: 12) (iii) The HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO: 13) (b) The light chain includes HVR-L1, HVR-L2 and HVR-L3, among which: (i) HVR-L1 sequence is RASQX4 X5 X6 TX7 X8 A (SEQ ID NO: 18) (ii) HVR-L2 sequence is SASX9 LX10 S; and (SEQ ID NO: 19) (iii) HVR-L3 sequence is QQX11 X12 X13 X14 PX15 T; (SEQ ID NO: 20) Among other things: X1 Is D or G; X2 Is S or L; X3 Is T or S; X4 Is D or V; X5 Is V or I; X6 Is S or N; X7 Is A or F; X8 Is V or L; X9 Is F or T; X10 Is Y or A; X11 Is Y, G, F or S; X12 Is L, Y, F or W; X13 Y, N, A, T, G, F or I; X14 Is H, V, P, T or I; X15 It is A, W, R, P or T. In a particular aspect, X1 Is D; X2 S and X3 Is T. In another aspect, X4 Is D; X5 Is V; X6 Is S; X7 Is A; X8 Is V; X9 Is F; X10 Is Y; X11 Is Y; X12 Is L; X13 Is Y; X14 Is H; X15 Is A. In another aspect, X1 Is D; X2 S and X3 Is T; X4 Is D; X5 Is V; X6 Is S; X7 Is A; X8 Is V; X9 Is F; X10 Is Y; X11 Is Y; X12 Is L; X13 Is Y; X14 Is H and X15 Is A. In another aspect, the heavy chain variable region includes one or more in (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-( HVR-H3)-(HC-FR4) has a framework sequence juxtaposed between the HVRs, and the light chain variable region contains one or more in (LC-FR1)-(HVR-L1)-(LC-FR2)- (HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4) The parallel framework sequence between HVR. In another aspect, the framework sequence is derived from a human common framework sequence. In another aspect, the heavy chain framework sequence is derived from Kabat subgroup I, II or III sequence. In another aspect, the heavy chain framework sequence is the VH subgroup III common framework. In another aspect, the one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 17). In another aspect, the light chain framework sequence is derived from the Kabat κ I, II, II, or IV subgroup sequence. In another aspect, the light chain framework sequence is the VL κ I common framework. In another aspect, the one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 24). In another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In another aspect, the murine constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, the minimal effector function is produced by "no effect Fc mutation" or non-glycosylation. In another example, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In another embodiment, an anti-PDL1 antibody is provided, which comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences, which have GFTFSDSWIH (SEQ ID NO: 25), AWISPYGGSTYYADSVKG (SEQ ID NO: 26) and RHWPGGFDY (SEQ ID NO: 13), respectively At least 85% sequence identity, or (b) The light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences, which have RASQDVSTAVA (SEQ ID NO: 27), SASFLYS (SEQ ID NO: 28) and QQYLYHPAT (SEQ ID NO: 29), respectively At least 85% sequence identity. In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region includes one or more in (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-( HVR-H3)-(HC-FR4) has a framework sequence juxtaposed between the HVRs, and the light chain variable region contains one or more in (LC-FR1)-(HVR-L1)-(LC-FR2)- (HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4) The parallel framework sequence between HVR. In another aspect, the framework sequence is derived from a human common framework sequence. In another aspect, the heavy chain framework sequence is derived from Kabat subgroup I, II or III sequence. In another aspect, the heavy chain framework sequence is the VH subgroup III common framework. In another aspect, the one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 17). In another aspect, the light chain framework sequence is derived from the Kabat κ I, II, II, or IV subgroup sequence. In another aspect, the light chain framework sequence is the VL κ I common framework. In another aspect, the one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 24). In another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In another aspect, the murine constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, the minimal effector function is produced by "no effect Fc mutation" or non-glycosylation. In another example, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In another embodiment, an isolated anti-PDL1 antibody is provided, which comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain sequence has at least 85% sequence identity with the heavy chain sequence: EVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 30), or (b) Light chain sequence and light chain sequence: DIQMTQSPSSLSASVGDRVTITCRA SQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 31) has at least 85% sequence identity. In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region includes one or more in (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-( HVR-H3)-(HC-FR4) has a framework sequence juxtaposed between the HVRs, and the light chain variable region contains one or more in (LC-FR1)-(HVR-L1)-(LC-FR2)- (HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4) The parallel framework sequence between HVR. In another aspect, the framework sequence is derived from a human common framework sequence. In another aspect, the heavy chain framework sequence is derived from Kabat subgroup I, II or III sequence. In another aspect, the heavy chain framework sequence is the VH subgroup III common framework. In another aspect, the one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 WGQGTLVTVSA (SEQ ID NO: 17). In another aspect, the light chain framework sequence is derived from the Kabat κ I, II, II, or IV subgroup sequence. In another aspect, the light chain framework sequence is the VL κ I common framework. In another aspect, the one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 24). In another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In another aspect, the murine constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, the minimal effector function is produced in prokaryotic cells. In another specific aspect, the minimal effector function is produced by "no effect Fc mutation" or non-glycosylation. In another example, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In another embodiment, an isolated anti-PDL1 antibody is provided, which comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain sequence has at least 85% sequence identity with the heavy chain sequence: EVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32), or (b) Light chain sequence and light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQD VSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 31) has at least 85% sequence identity. In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region includes one or more in (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-( HVR-H3)-(HC-FR4) has a framework sequence juxtaposed between the HVRs, and the light chain variable region contains one or more in (LC-FR1)-(HVR-L1)-(LC-FR2)- (HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4) The parallel framework sequence between HVR. In another aspect, the framework sequence is derived from a human common framework sequence. In another aspect, the heavy chain framework sequence is derived from Kabat subgroup I, II or III sequence. In another aspect, the heavy chain framework sequence is the VH subgroup III common framework. In another aspect, the one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 14) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 15) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 WGQGTLVTVSS (SEQ ID NO: 33). In another aspect, the light chain framework sequence is derived from the Kabat κ I, II, II, or IV subgroup sequence. In another aspect, the light chain framework sequence is the VL κ I common framework. In another aspect, the one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 24). In another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In another aspect, the murine constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, the minimal effector function is produced in prokaryotic cells. In another specific aspect, the minimal effector function is produced by "no effect Fc mutation" or non-glycosylation. In another example, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In another aspect, the heavy chain variable region includes one or more in (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-( HVR-H3)-(HC-FR4) has a framework sequence juxtaposed between the HVRs, and the light chain variable region contains one or more in (LC-FR1)-(HVR-L1)-(LC-FR2)- (HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4) The parallel framework sequence between HVR. In another aspect, the framework sequence is derived from a human common framework sequence. In another aspect, the heavy chain framework sequence is derived from Kabat subgroup I, II or III sequence. In another aspect, the heavy chain framework sequence is the VH subgroup III common framework. In another aspect, the one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 34) HC-FR2 WVRQAPGKGLEWVA (SEQ ID NO: 35) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 WGQGTLVTVSS (SEQ ID NO: 33). In another aspect, the light chain framework sequence is derived from the Kabat κ I, II, II, or IV subgroup sequence. In another aspect, the light chain framework sequence is the VL κ I common framework. In another aspect, the one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23) LC-FR4 FGQGTKVEIK (SEQ ID NO: 36). In another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In another aspect, the murine constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, the minimal effector function is produced by "no effect Fc mutation" or non-glycosylation. In another example, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In another embodiment, an anti-PDL1 antibody is provided, which comprises heavy chain and light chain variable region sequences, wherein: (c) The heavy chain further includes HVR-H1, HVR-H2, and HVR-H3 sequences, which have GFTFSDSWIH (SEQ ID NO: 4), AWISPYGGSTYYADSVKG (SEQ ID NO: 5) and RHWPGGFDY (SEQ ID NO: 6), respectively At least 85% sequence identity, or (d) The light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences, which have RASQDVSTAVA (SEQ ID NO: 1), SASFLYS (SEQ ID NO: 2) and QQYLYHPAT (SEQ ID NO: 3), respectively At least 85% sequence identity. In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain variable region includes one or more in (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-( HVR-H3)-(HC-FR4) has a framework sequence juxtaposed between the HVRs, and the light chain variable region contains one or more in (LC-FR1)-(HVR-L1)-(LC-FR2)- (HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4) The parallel framework sequence between HVR. In another aspect, the framework sequence is derived from the human common framework sequence. In another aspect, the heavy chain framework sequence is derived from Kabat subgroup I, II or III sequence. In another aspect, the heavy chain framework sequence is the VH subgroup III common framework. In another aspect, the one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 34) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 35) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 16) HC-FR4 WGQGTLVTVSSASTK (SEQ ID NO: 33). In another aspect, the light chain framework sequence is derived from the Kabat κ I, II, II, or IV subgroup sequence. In another aspect, the light chain framework sequence is the VL κ I common framework. In another aspect, the one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 21) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 22) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 23) LC-FR4 FGQGTKVEIKR (SEQ ID NO: 24). In another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In another specific aspect, the human constant region is IgG1. In another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In another aspect, the murine constant region is IgG2A. In another specific aspect, the antibody has reduced or minimal effector function. In another specific aspect, the minimal effect function is produced by "no effect Fc mutation" or non-glycosylation. In another example, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In another embodiment, an isolated anti-PDL1 antibody is provided, which comprises heavy chain and light chain variable region sequences, wherein: (a) The heavy chain sequence has at least 85% sequence identity with the heavy chain sequence: EVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 8), or (b) Light chain sequence and light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQ DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 7) has at least 85% sequence identity. In some embodiments, an isolated anti-PDL1 antibody comprising heavy chain and light chain variable region sequences is provided, wherein the light chain variable region sequence and the amino acid sequence of SEQ ID NO: 7 have at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % Sequence identity. In some embodiments, an isolated anti-PDL1 antibody comprising heavy chain and light chain variable region sequences is provided, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, and SEQ ID NO: 8 At least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity . In some embodiments, an isolated anti-PDL1 antibody comprising heavy chain and light chain variable region sequences is provided, wherein the light chain variable region sequence has the same amino acid sequence as SEQ ID NO: 7 at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % Sequence identity and the heavy chain variable region sequence has the amino acid sequence SEQ ID NO: 8 at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In another embodiment, an isolated anti-PDL1 antibody comprising heavy chain and light chain sequences is provided, wherein: (A) heavy chain sequence and a heavy chain having the sequence: EVQLVESGGGLVQPGGSLRL SCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10) 85% sequence identity at least, or (B) a light chain sequence and light chain sequence: DIQMTQSPSSLSASVGDRVTI TCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9) at least 85% sequence identity. In some embodiments, an isolated anti-PDL1 antibody comprising heavy chain and light chain sequences is provided, wherein the light chain sequence and the amino acid sequence of SEQ ID NO: 9 have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, an isolated anti-PDL1 antibody comprising a heavy chain and a light chain sequence is provided, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89% of SEQ ID NO: 10 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, an isolated anti-PDL1 antibody comprising a heavy chain and a light chain sequence is provided, wherein the light chain sequence has the same amino acid sequence as SEQ ID NO: 9 at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and heavy The chain sequence has an amino acid sequence SEQ ID NO: 10 at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the isolated anti-PDL1 antibody is an oxidized monoclonal antibody. In some embodiments, the oxidized monoclonal antibody in the formulation comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the oxidized monoclonal antibody in the formulation comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10, wherein one or more of W33, W50, or W101 is oxidized. In some embodiments, the oxidized monoclonal antibody in the formulation comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10, wherein one or more of M253 and M429 are oxidized. In some embodiments, the oxidized monoclonal antibody retains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. % Biological activity (such as binding to the target or therapeutic efficacy) before storage (that is, when the pharmaceutical formulation is prepared). In some embodiments, the isolated anti-PDL1 antibody is a glycosylated monoclonal antibody. In some embodiments, the glycosylated monoclonal antibody in the formulation comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the glycosylated monoclonal antibody in the formulation comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10, in which one or more lysine acids are glycosylated. In some embodiments, the glycosylated monoclonal antibody in the formulation comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10, wherein K65 is glycosylated. In some embodiments, the isolated anti-PDL1 antibody is deglycosylated. In any embodiment herein, the isolated anti-PDL1 antibody can bind to human PD-L1, such as the human PD-L1 shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In another embodiment, an isolated nucleic acid encoding any of the antibodies described herein is provided. In some embodiments, the nucleic acid further comprises a nucleic acid vector suitable for expressing any of the aforementioned anti-PDL1 antibodies. In another specific aspect, the vector is in a host cell suitable for expression of nucleic acid. In another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In another specific aspect, the eukaryotic cells are mammalian cells, such as Chinese Hamster Ovary (CHO). Antibodies or antigen-binding fragments thereof can be produced using methods known in the art (for example, by methods comprising culturing host cells containing nucleic acid encoding any of the aforementioned anti-PDL1 antibodies or antigen-binding fragments). Such antibodies or fragments are prepared in a form suitable for expression under the conditions for recovering the antibodies or fragments.B. Antibody preparation Exemplary methods for preparing antibodies in formulations using techniques available for antibody production in this technology are described in more detail in the following sections. The anti-system is directed against the antigen of interest (ie PD-L1, such as human PD-L1). The antigen is preferably a biologically important polypeptide, and administering the antibody to a mammal suffering from a disorder can produce therapeutic benefits in that mammal. (i) Antigen preparation Optionally, soluble antigens or fragments thereof that bind to other molecules can be used as immunogens for antibody production. For transmembrane molecules (such as receptors), fragments thereof (such as the extracellular domain of receptors) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from natural sources (such as cancer cell lines) or may be cells that have been transformed by recombinant technology to express transmembrane molecules. Other antigens and their forms suitable for preparing antibodies will be obvious to those familiar with this technology. (ii) Certain antibody-based methods Multiple antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of relevant antigens and adjuvants. It can be used to bind related antigens to proteins that are immunogenic for the species to be immunized, such as keyhole cyanogenin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using bifunctional or derivatized agents Carry out, for example, maleimide, benzyl sulfosuccinimide (by cysteine residues), N-hydroxysuccinimide (by lysine residues), glutaraldehyde , Succinic anhydride, SOCl2 Or R1 N=C=NR, where R and R1 Different alkyl groups. By combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and intradermal injection of the solution at multiple sites, the animal is exposed to antigen, Immunogenic conjugate or derivative immunization. One month later, the animals were injected subcutaneously with 1/5 to 1/10 of the original amount of the peptide or conjugate in Freund's complete adjuvant at multiple sites. After 7 to 14 days, the animals were bled and the serum antibody titer was analyzed. Beat the animal until the price of power stabilizes. Preferably, the animal is beaten with a conjugate of the same antigen (but the antigen is bound to different proteins and/or through different cross-linking agents). Conjugates can also be produced as protein fusions in recombinant cell culture. Likewise, aggregating agents such as alum are suitable for enhancing the immune response. The monoclonal antibody of the present invention can use the fusion tumor method for the first time by Kohler et al.Nature , 256:495 (1975), and further described in, for example, Hongo et al. on human-human fusion tumors,Hybridoma , 14 (3): 253-260 (1995); Harlow et al.,Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd edition. 1988); Hammerling et al.,Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981); and Ni,Xiandai Mianyixue , 26(4):265-268 (2006) in the method of fusion tumor manufacturing. Other methods include, for example, the methods described in U.S. Patent No. 7,189,826 regarding the production of single human natural IgM antibodies from fusion tumor cell lines. Human fusion tumor technology (Trioma technology) also describes Vollmers and Brandlein,Histology and Histopathology , 20(3):927-937 (2005) and Vollmers and Brandlein,Methods and Findings in Experimental and Clinical Pharmacology , 27(3):185-91 (2005). For various other fusion tumor technologies, see, for example, US 2006/258841; US 2006/183887 (fully human antibodies); US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and US Patent Nos. 7,078,492 and No. 7,153,507. An exemplary protocol for the production of monoclonal antibodies using the fusion tumor method is described below. In one embodiment, mice or other suitable host animals (such as hamsters) are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. By multiple subcutaneous (sc) or intraperitoneal (ip) injections of the polypeptide of the present invention or its fragments and adjuvants (such as monophosphoryl lipid A (MPL)/trehalose dicyanomethicin (TDM) ( Ribi Immunochem. Research, Inc., Hamilton, Mont.)) and produce antibodies in animals. The polypeptides (eg, antigens) or fragments thereof of the present invention can be prepared using methods well known in the art (such as recombinant methods), some of which are further described herein. Analyze the anti-antigen antibodies in the serum from the immunized animals, and administer booster immunity as appropriate. Isolate lymphocytes from animals that produce anti-antigen antibodies. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent (such as polyethylene glycol) to form fusion tumor cells. See e.g. Goding,Monoclonal Antibodies: Principles and Practice , Page 59-Page 103 (Academic Press, 1986). It is possible to use myeloma cells that efficiently fuse, support stable and high-level antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Exemplary myeloma cells include, but are not limited to, murine myeloma cell lines, such as MOPC-21 and MPC-11 mouse tumors derived from Salk Institute Cell Distribution Center, San Diego, Calif. USA and those derived from American Type Culture Collection, Rockville, Md. USA SP-2 or X63-Ag8-653 cells. Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor,J. Immunol. , 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The fusion tumor cells thus prepared are seeded and grown in a suitable medium (for example, a medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium used for the fusion tumor will usually include hypoxanthine, aminopterin, and thymidine ( HAT medium), these substances will prevent the growth of HGPRT-deficient cells. Preferably, as for example Even et al.,Trends in Biotechnology , 24(3), 105-108 (2006), the use of serum-free fusion tumor cell culture method to reduce the use of animal-derived serum (such as fetal bovine serum). Oligopeptides as a tool to increase the production rate of fusion tumor cell cultures are described in FranekTrends in Monoclonal Antibody Research , 111-122 (2005). In particular, the standard medium is rich in certain amino acids (alanine, serine, aspartamide, proline) or protein hydrolysate fractions, and can be derived from three to six amino acid residues Synthetic oligopeptides composed of bases significantly inhibit cell death. The peptides are present in millimolar or higher concentrations. The production of monoclonal antibodies bound to the antibodies of the present invention can be analyzed in the medium in which the fusion tumor cells are grown. The binding specificity of monoclonal antibodies produced by fusion tumor cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined by, for example, Scatchard analysis. See, for example, Munson et al.,Anal. Biochem. , 107:220 (1980). After identifying the fusion tumor cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be sub-populated by restrictive dilution procedures and grown by standard methods. See, for example Goding, supra. Suitable media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, fusion tumor cells can be cultured as ascites tumors in animals in vivo. The monoclonal antibody system secreted by the sub-pure line is appropriately combined with the culture medium, by the conventional immunoglobulin purification procedures (such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography). Ascites fluid or serum separation. A procedure for separating proteins from fusion tumor cells is described in US 2005/176122 and US Patent No. 6,919,436. This method involves the use of minimal salts (such as easily soluble salts) in the binding process and preferably a small amount of organic solvents in the dissolution process. (iii) Certain library screening methods The antibodies of the present invention can be prepared by using combinatorial libraries to screen antibodies with desired activities. For example, various methods known in the art can be used to generate phage display libraries and screen such libraries for antibodies with desired binding characteristics. Such methods are generally described in Hoogenboom et al.Methods in Molecular Biology 178:1-37 (eds by O'Brien et al., Human Press, Totowa, NJ, 2001). For example, as Lee et al.,J. Mol. Biol. (2004), 340(5):1073-93, a method of generating antibodies of interest is by using phage antibody libraries. In general, synthetic antibody clones are selected by screening a phage library containing various fragments of the antibody variable region (Fv) fused to the phage sheath protein. Screen such phage libraries by performing affinity chromatography on the desired antigen. The pure lines of Fv fragments that appear to be capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding pure lines in the library. The binding agent is then eluted from the antigen and can be further enriched by additional antigen adsorption/dissolution cycles. Can select the phage clone of interest by designing a suitable antigen screening program, and then use the Fv sequence and Kabat from the phage clone of interestEt al, Sequences of Proteins of Immunological Interest , 5th edition, NIH Publication 91-3242, Bethesda MD (1991), Vol. 1-3 describes suitable constant region (Fc) sequences to construct a full-length antibody clone to obtain any antibody of the present invention. In certain embodiments, the antigen-binding domain of an antibody is formed by two variable (V) regions with about 110 amino acids, each from the light chain (VL) and the heavy chain (VH), both of which are Presenting three hypervariable loops (HVR) or complementarity determining regions (CDR). The variable domain can be as a single-chain Fv (scFv) fragment (wherein VH and VL are covalently linked via a flexible short peptide) or as a Fab fragment (wherein VH and VL are each fused to the constant domain and interact with non-covalent bonds) , Functionally displayed on phage, such as Winter et al.,Ann. Rev. Immunol. , 12: 433-455 (1994). As used herein, the pure line of phage encoding scFv and the pure line of phage encoding Fab are called "Fv pure line" or "Fv pure line". The VH and VL gene libraries can be separately selected by polymerase chain reaction (PCR), and randomly recombined in the phage library, and then the antigen-binding clones can be searched, such as Winter et al.,Ann. Rev. Immunol. , 12: 433-455 (1994). Libraries from immunized sources can provide high-affinity antibodies to immunogens without constructing fusion tumors. Alternatively, the natural library can be selected to provide a single source of human antibodies to a wide range of non-self antigens and self-antigens without any immune function, such as Griffiths et al.,EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be synthesized by selecting unrearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode hypervariable CDR3 regions and complete in vitro rearrangement, such as Hoogenboom and Winter,J. Mol. Biol. , 227: 381-388 (1992). In certain embodiments, filamentous phages are used to display antibody fragments by fusion to the minor sheath protein pill. These antibody fragments can be presented as single-chain Fv fragments, in which the VH domain and the VL domain are connected to the same polypeptide chain via a flexible polypeptide spacer, such as Marks et al.J. Mol. Biol. , 222:581-597 (1991); or presented as a Fab fragment in which one chain is fused with pIII and the other chain is secreted into the periplasm of bacterial host cells, where it is replaced by wild-type outer coat A part of the protein assembles the Fab-coating protein structure presented on the surface of the phage, such as Hoogenboom et al.Nucl. Acids Res., 19 : 4133-4137 (1991). In general, nucleic acids encoding antibody gene fragments can be obtained from immune cells collected from humans or animals. If an anti-antigen pure line library is needed, the individual can be immunized with antigen to produce an antibody response, and splenocytes and/or circulating B cells and other peripheral blood lymphocytes (PBL) can be recovered for library construction. In one embodiment, the human antibody gene fragment library that focuses on the anti-antigen pure line is obtained by transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) Anti-antigen antibody response is generated in the medium so that B cells can produce human antibodies against antigen by antigen immunization. The production of transgenic mice producing human antibodies will be described below. B cells expressing antigen-specific membrane-bound antibodies can be isolated by using suitable screening procedures (e.g., by separating cells by antigen affinity chromatography or by adsorbing cells to fluorescent dye-labeled antigens followed by flow-activated cell sorting ( FACS)) to obtain additional enrichment of the anti-antigen reactive cell population. Alternatively, use spleen cells and/or B cells or other PBL from unimmunized donors to better present the possible antibody profile, and it is also allowed to use any animal (human or non-human) species whose antigen is not antigenic. Build an antibody library. For libraries incorporating in vitro antibody gene construction, stem cells are collected from individuals to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as humans, mice, rats, rabbits, wolves, canines, cats, pigs, cows, horses, and bird species. The nucleic acid encoding antibody variable gene segments (including VH and VL segments) is recovered from the cell of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes, reusing primers matching the 5'and 3'ends of the rearranged VH and VL genes, and by Obtained by polymerase chain reaction (PCR), such as Orlandi et al.,Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989) to obtain various V gene libraries for performance. The V gene can be amplified from cDNA and genomic DNA using the backward primer at the 5'end of the exon outside the mature V-domain and the forward primer located inside the J-segment, as shown in Orlandi et al. (1989) And Ward et al.,Nature , 341:544-546 (1989). However, for self-cDNA amplification, the backward primer can also be positioned in the leading exon, such as Jones et al.Biotechnol. , 9:88-89 (1991), and the forward primer can also be located inside the constant region, such as Sastry et al.,Proc. Natl. Acad. Sci. (USA) , 86:5728-5732 (1989). To maximize complementarity, degeneracy can be integrated into these primers, as described in Orlandi et al. (1989) or Sastry et al. (1989). In some embodiments, library diversity can be maximized by using PCR primers targeting each V-gene family, so as to increase all available VH and VL arrangements present in immune cell nucleic acid samples, such as Marks Et al,J. Mol. Biol. , 222:581-597 (1991), or as described in Orum et al.,Nucleic Acids Res. , 21:4491-4498 (1993) described in the method. For the selection of amplified DNA into the expression vector, rare restriction sites can be introduced into the PCR primer as a marker at one end, as described in Orlandi et al. (1989); or the marker primer can be used for other PCR amplification. Increase, as Clackson et al.,Nature , 352: 624-628 (1991). The synthetically rearranged V gene library can be obtained from V gene segments in vitro. Most of the human VH-gene segments have been selected and sequenced (reported in Tomlinson et al.,J. Mol. Biol. , 227:776-798 (1992)), and a map has been drawn (reported in Matsuda et al.,Nature Genet. ,3 :88-94 (1993)); these cloning segments (including all major configurations of H1 and H2 loops) and PCR primers encoding H3 loops of various sequences and lengths can be used to generate various VH gene libraries, such as Hoogenboom And Winter,J. Mol. Biol. , 227:381-388 (1992). It is also possible to obtain a VH library with all sequence diversity concentrated in the long H3 loop of the single-fold length, such as Barbas et al.,Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human Vκ and Vλ segments have been selected and sequenced (reported in Williams and Winter,Eur. J. Immunol. , 23:1456-1461 (1993)) and can be used to obtain synthetic light chain libraries. Based on the synthesis of a certain range of VH and VL folds and L3 and H3 lengths, the V gene library will encode antibodies with a large amount of structural diversity. After amplifying the V-gene encoding DNA, the germline V-gene segment can be based on Hoogenboom and Winter,J. Mol. Biol. , 227: 381-388 (1992) method of in vitro rearrangement. Several methods can be used to construct antibody fragment libraries by combining VH and VL gene libraries. Each library can be produced in different vectors, and the vectors are recombined in vitro (e.g. Hogrefe et al.,Gene , 128: 119-126 (1993)) or recombination in vivo by combined infection (e.g. Waterhouse et al.,Nucl. Acids Res. , 21: 2265-2266 (1993) described in loxP system). In vivo recombination methods use the double-stranded nature of Fab fragments to overcome the limitation of library size imposed by E. coli transformation power. The native VH and VL libraries were cloned separately, one was cloned into a phagemid and the other was cloned into a phage vector. Then the two libraries were combined by phage infection of bacteria containing phagemid, so that each cell contained a different combination and the size of the library was only affected by the number of cells present (about 1012 A pure line) restriction. Both vectors contain in vivo recombination signals, so that VH and VL genes are recombined on a single replicon and co-packaged in phage virus particles. These large libraries provide a large amount of good affinity (Kd -1 For about 10-8 M) Multiple antibodies. Alternatively, the libraries can be sequentially selected and cloned into the same vector, for example, as Barbas et al.,Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); or assembled together by PCR and then colonized, for example, as Clackson et al.,Nature, 352:624-628 (1991). PCR assembly can also be used to ligate VH and VL DNA with DNA encoding a flexible peptide spacer to form a single-chain Fv (scFv) library. In another technique, "in cell PCR assembly" is used to combine VH and VL genes by PCR in lymphocytes, and then as Embleton et al.,Nucl. Acids Res. , 20: 3831-3837 (1992), a library of clonal linkage genes. Antibodies (natural or synthetic) produced by the native library can have moderate affinity (about 106 To 107 M-1 K ofd -1 ), but affinity maturation can also be simulated by constructing a second library and selecting from it, as described in Winter et al. (1994) (supra). For example, by using error-prone polymerases (reported in Leung et al., Technique 1: 11-15 (1989)) Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) Method or Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992), introduce mutations at random locations in vitro. In addition, affinity maturation can be performed by randomly mutating one or more CDRs in the selected individual Fv clones using PCR with primers carrying random sequences spanning the CDRs of interest, and screening higher affinity clones for example. WO 9607754 (published on March 14, 1996) describes a method for inducing mutations in the complementarity determining region of an immunoglobulin light chain to form a light chain gene library. Another effective method is to recombine the selected VH domain or VL domain by phage display with a library of naturally occurring V domain variants obtained from unimmunized donors and use several rounds of chain shuffling to screen for higher affinity, such as Marks et al. ,Biotechnol. , 10:779-783 (1992). This technique makes it possible to produce an affinity of about 10-9 M or 10-9 Antibodies and antibody fragments below M. The library can be screened by various techniques known in the art. For example, antigen can be used to coat the wells of the adsorption plate, which can be displayed on the host cells attached to the adsorption plate or used for cell sorting, or bound to biotin for capture by streptavidin-coated beads , Or used in any other method of panning phage display libraries. The sample of the phage library is contacted with the immobilized antigen under conditions suitable for binding at least a portion of the phage particle to the adsorbent. Generally speaking, conditions including pH, ionic strength, temperature and the like are selected to simulate physiological conditions. The phage bound to the solid phase is washed and then lysed by acid, for example, as in Barbas et al.,Proc. Natl. Acad. Sci USA , 88:7978-7982 (1991); or by alkali dissolution, such as Marks et al.J. Mol. Biol. , 222:581-597 (1991); or by antigen competition (for example, with Clackson et al.,Nature , 352:624-628 (1991), a similar procedure to the Antigen Competition Law). The phage can be enriched 20-1,000 times in a single round of selection. In addition, the concentrated phage can be grown in bacterial culture and undergo another round of selection. The selection efficiency depends on many factors, including the dissociation kinetics during washing and whether multiple antibody fragments on a single phage can simultaneously engage with the antigen. Antibodies with fast dissociation kinetics (and weak binding affinity) can be retained by using short-time washing, multivalent phage display, and high coating density of antigen in the solid phase. The high density not only stabilizes the phage through multivalent interaction, but also facilitates the recombination of the dissociated phage. Antibodies with slow dissociation kinetics (and good binding affinity) can be selected by using long-term washing and monovalent phage display (such as Bass et al.,Proteins , 8: 309-314 (1990) and WO 92/09690) and low antigen coating density (as described in Marks et al., Biotechnol., 10: 779-783 (1992)) promoted. It is possible to choose between phage antibodies with different or even slightly different affinities for the antigen. However, random mutations of the selected antibody (for example, as performed in some affinity maturation techniques) may produce many mutants, most of which bind to the antigen and a few have higher affinity. The use of restricted antigens can compete to eliminate very few high-affinity phages. In order to retain all the higher affinity mutants, the phage can be incubated with an excess of biotin-labeled antigen, but the molar concentration of the biotin-labeled antigen is lower than the antigen's target molar affinity constant. The high-affinity bound phage can then be captured by paramagnetic beads coated with streptavidin. This type of "balanced trap" allows antibodies to be selected based on their binding affinity, and its sensitivity allows the isolation of mutant clones with only twice the high affinity from a significant excess of phages with lower affinity. The conditions used in washing and solid phase-bound phage can also be manipulated to distinguish based on dissociation kinetics. Anti-antigen clones can be selected based on activity. In certain embodiments, the present invention provides anti-antigen antibodies that bind to living cells that naturally express antigens or to free-floating antigens or antigens linked to other cellular structures. Fv clones corresponding to such anti-antigen antibodies can be selected by the following steps: (1) The anti-antigen clones are isolated from the phage library as described above, and the population is expanded by cultivating the population in a suitable bacterial host as appropriate. Enhance the isolated population of the pure line of phage; (2) select the antigen and the second protein for blocking activity and non-blocking activity respectively; (3) adsorb the pure line of anti-antigen phage to the fixed antigen; (4) use excessive The second protein dissociates any undesired pure lines that recognize the antigen binding determinants that overlap or share the binding determinants of the second protein; and (5) the pure lines that are still adsorbed after the dissociation step (4) is completed. Depending on the circumstances, the pure line with the desired blocking/non-blocking characteristics can be further enriched by repeating the selection procedure described herein one or more times. It is easy to isolate the DNA encoding the fusion tumor-derived monoclonal antibody or phage displaying Fv pure line of the present invention, and use conventional procedures (for example, by using a DNA template designed to specifically amplify the relevant heavy chain from the fusion tumor or phage and Oligonucleotide primers in the light chain coding region) for sequencing. After isolation, the DNA can be placed in the expression vector, and then the expression vector can be transfected into host cells that do not otherwise produce immunoglobulin (such as E. coli cells, ape COS cells, Chinese hamster ovary (CHO) cells or myeloma cells ) To obtain the synthesis of the desired monoclonal antibody in the recombinant host cell. Commentary articles on the recombinant expression of DNA encoding antibodies in bacteria include Skerra et al.,Curr. Opinion in Immunol. , 5: 256 (1993) and Pluckthun,Immunol. Revs , 130: 151 (1992). The DNA encoding the Fv clone of the present invention can be combined with known DNA sequences encoding heavy and/or light chain constant regions (for example, appropriate DNA sequences can be obtained from Kabat et al. (supra)) to form a full-length or partial-length heavy chain And/or the pure line of light chain. It should be understood that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. Fv clones derived from the variable domain DNA of one animal (such as human) species and subsequently fused with the constant region DNA of another animal species to form a "hybrid" full-length heavy chain and/or light chain coding sequence are included as used herein In the definition of "chimeric" and "hybrid" antibodies. In certain embodiments, the Fv clone derived from human variable DNA is fused to human constant region DNA to form a full-length or partial-length human heavy chain and/or light chain coding sequence. The DNA encoding the anti-antigen antibody obtained from the hybridoma of the present invention can also be modified, for example, using human heavy chain constant domain and light chain constant domain coding sequences to replace homologous murine sequences derived from pure hybridoma lines (for example, using Morrison et al.,Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984) method). The DNA encoding the antibody or fragment derived from the fusion tumor or Fv clone can be further modified by covalently joining all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, "chimeric" or "hybrid" antibodies with the binding specificity of the Fv pure line of the present invention or the binding specificity of the antibody derived from the hybridoma line can be prepared. (iv) Humanization and human antibodies Various methods of humanizing non-human antibodies are known in the art. For example, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from the "import" variable domain. Humanization can basically follow the methods of Winter and collaborators (Jones et al.,Nature , 321:522-525 (1986); Riechmann et al.,Nature , 332:323-327 (1988); Verhoeyen et al.,Science , 239:1534-1536 (1988)), by substituting rodent CDR or CDR sequences for the corresponding sequences of human antibodies. Therefore, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which variable domains that are substantially less than the complete human variable domain are replaced with corresponding sequences from non-human species. In fact, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues have been substituted with residues from similar sites in rodent antibodies. The alternatives (light chain and heavy chain) of human variable domains to be used in the production of humanized antibodies are extremely important for reducing antigenicity. According to the so-called "best fit" method, the variable domain sequence of rodent antibodies is screened against the entire library of known human variable domain sequences. Then, accept the human sequence closest to the rodent sequence as the human framework (FR) of the humanized antibody (see Sims et al.,J. Immunol ., 151:2296 (1993); Chothia et al.,J. Mol. Biol ., 196:901 (1987)). Another method uses a specific framework derived from the common sequence of all human antibodies with a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al.,Proc. Natl. Acad Sci. USA , 89:4285 (1992); Presta et al.,J. Immunol. , 151:2623 (1993)). More importantly, the antibody is humanized and retains high affinity for the antigen and other beneficial biological properties. To achieve this goal, according to an embodiment of the method, a humanized antibody is prepared by using a three-dimensional model of the parental sequence and the humanized sequence to analyze the parental sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and familiar to those familiar with the technology. A computer program can be obtained that illustrates and presents the possible three-dimensional configuration of the selected candidate immunoglobulin sequence. The examination of these presentations allows the analysis of the possible role of residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined with the recipient and import sequences in order to obtain desired antibody characteristics, such as increased affinity for the target antigen. In general, the residues in the hypervariable region are directly and most substantially involved in affecting antigen binding. The human antibody of the present invention can be constructed by combining Fv cloned variable domain sequences selected from a human-derived phage display library and known human constant domain sequences as described above. Alternatively, the human monoclonal antibody of the present invention can be prepared by the fusion tumor method. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been developed, for example, by KozborJ. Immunol. , 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications , Page 51-page 63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al.,J. Immunol. , 147: 86 (1991) description. It is possible to produce transgenic animals (such as mice) that can produce a full library of human antibodies once immunized in the absence of endogenous immunoglobulin production. For example, the antibody heavy chain junction region in chimeric and germline mutant mice has been described (JH ) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. The transfer of human germline immunoglobulin gene arrays to such germline mutant mice will cause the production of human antibodies after antigen challenge. See, for example, Jakobovits et al.,Proc. Natl. Acad. Sci. USA , 90:2551 (1993); Jakobovits et al.,Nature , 362:255-258 (1993); Bruggermann et al.,Year in Immuno. , 7:33 (1993); and Duchosal et al.,Nature 355:258 (1992). Gene shuffling can also be used to obtain human antibodies from non-human (e.g., rodent) antibodies, where the human antibody has similar affinity and specificity to the original non-human antibody. According to this method, also known as "epitope imprinting", the variable region of the heavy or light chain of the non-human antibody fragment obtained by the phage display technology as described herein is replaced with a human V domain gene library to form a non-human chain /Human chain scFv or Fab chimera population. The use of antigen for selection can lead to the separation of non-human chain/human chain chimeric scFv or Fab, where the human chain recovers and removes the damaged antigen binding site when the primary phage presents the corresponding non-human chain in the clone, that is, epitope determination (Impression) The choice of human chain collocation. When this method is repeated to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published on April 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technology provides complete human antibodies that do not have FR or CDR residues of non-human origin. (v) Antibody fragments Antibody fragments can be produced by traditional methods (such as enzymatic digestion) or by recombinant techniques. In some cases, it is advantageous to use antibody fragments instead of whole antibodies. The smaller size fragments allow rapid clearance and can lead to improved entry into solid tumors. For reviews of certain antibody fragments, see Hudson et al. (2003)Nat. Med. 9:129-134. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments can be obtained by proteolytic digestion of whole antibodies (see, for example, Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al.,Science , 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in E. coli and secreted from E. coli, thus allowing easy production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 Fragment (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another method, F(ab')2 Fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') with prolonged half-life in vivo and containing rescue receptor binding epitope residues2 Fragments are described in US Patent No. 5,869,046. Skilled practitioners will obviously know other manufacturing techniques for antibody fragments. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185, U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only substances that have a complete binding site without a constant region; therefore, they can be adapted to reduce non-specific binding during use in vivo. The scFv fusion protein can be constructed so that the effector protein is fused to the amine or carboxyl end of the scFv. SeeAntibody Engineering , Edited by Borrebaeck, same as above. Antibody fragments may also be "linear antibodies" as described in, for example, US Patent No. 5,641,870. Such linear antibodies can be monospecific or bispecific antibodies. (vi) Multispecific antibodies Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes are usually derived from different antigens. Although such molecules usually only bind two different epitopes (ie bispecific antibodies, BsAb), antibodies with additional specificities (such as trispecific antibodies) when used herein are covered by this expression . Bispecific antibodies in the form of full-length antibodies or antibody fragments can be prepared (e.g. F(ab')2 Bispecific antibodies). Methods of preparing bispecific antibodies are known in the art. Traditionally, the production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al.,Nature , 305:537-539 (1983)). Due to the random allocation of immunoglobulin heavy and light chains, these fusion tumors (quaternary fusion tumors) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Generally, the purification of the correct molecule by affinity chromatography steps is quite troublesome and the yield is low. Similar procedures are disclosed in WO 93/08829 and Traunecker et al.,EMBO J. , 10:3655-3659 (1991). According to different methods, the antibody variable domain (antibody-antigen binding site) with the desired binding specificity is fused to the immunoglobulin constant domain sequence. The fusion is preferably a fusion with an immunoglobulin heavy chain constant domain comprising at least part of the hinge region, CH2 and CH3 regions. Usually the first heavy chain constant region (CH1) present in at least one of these fusions contains the necessary sites for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. When the unequal ratios of the three polypeptide chains used in the construction provide the best yield, this provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in the embodiment. However, when the performance of at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into one expression vector. In one embodiment of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (provided The second binding specificity) composition. It has been found that because the immunoglobulin light chain is only present in half of the bispecific molecule to provide a convenient way of separation, this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain combination. This method is disclosed in WO 94/04690. For other details on the production of bispecific antibodies, see, for example, Suresh et al.,Methods in Enzymology , 121:210 (1986). According to another method described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. One interface contains antibody constant domain CH 3 At least part of the domain. In this way, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (such as tyrosine or tryptophan). Complementary "cavities" that are the same or similar in size to the large side chain are formed on the interface of the second antibody molecule by replacing the side chain of a large amino acid with a side chain of a smaller amino acid (such as alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other undesirable end products (such as homodimers). Bispecific antibodies include cross-linked or "heterologous binding" antibodies. For example, one of the antibodies in the form of a heteroconjugate can be coupled to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to undesirable cells (US Patent No. 4,676,980) and used to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents and many crosslinking techniques are well known in the art and are disclosed in US Patent No. 4,676,980. Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al.,Science , 229:81 (1985) describes a kind of intact antibody can produce F(ab') by protein cleavage2 Fragment program. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide bond formation. The Fab' fragments produced are then converted into thionitrobenzoate (TNB) derivatives. Then one Fab'-TNB derivative is converted into Fab'-thiol by reduction with mercaptoethylamine, and it is mixed with an equal molar amount of another Fab'-TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as reagents for the selective immobilization of enzymes. Recent developments have promoted the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al.,J. Exp. Med. , 175:217-225 (1992) describes a fully humanized bispecific antibody F(ab')2 Preparation of molecules. Each Fab' fragment is separately secreted from E. coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al.,J. Immunol., 148 (5):1547-1553 (1992). The leucine zipper peptides from Fos and Jun proteins were linked to the Fab' parts of two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, and then it is reoxidized to form an antibody heterodimer. This method can also be used to produce antibody homodimers. Hollinger et al.,Proc. Natl. Acad. Sci. USA, The "bifunctional antibody" technology described in 90:6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. These fragments comprise the light chain variable domain (VL ) Of the heavy chain variable domain (VH ), the linker is too short to allow pairing between the two domains on the same chain. Therefore, forcing a segment of VH And VL Complementation of domain and another segment VL And VH The domains pair, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol. , 152:5368 (1994). Antibodies with more than two valences are also considered. For example, trispecific antibodies can be prepared.Tuft Et al. J. Immunol. 147: 60 (1991). (vii) Single domain antibody In some embodiments, the antibodies of the invention are single domain antibodies. A single domain antibody is a single polypeptide chain that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, for example, US Patent No. 6,248,516 B1). In one embodiment, a single domain antibody consists of all or part of the heavy chain variable domain of the antibody. (viii) Antibody variants In some embodiments, modifications to the amino acid sequence of the antibodies described herein are encompassed. For example, it may be necessary to improve the binding affinity and/or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and/or insertion and/or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final structure, and the limitation is that the final structure has the required characteristics. The amino acid changes can be introduced into the target antibody amino acid sequence when the sequence is made. (ix) Antibody derivatives The antibodies of the present invention may be further modified to contain additional non-protein moieties known in the art and readily available. In certain embodiments, the portion suitable for derivatizing the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include (but are not limited to) polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone , Poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) And dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylene polyol (such as glycerin), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, the polymers can be the same or different molecules. Generally speaking, the number and/or type of polymers used for derivatization can be determined based on considerations including (but not limited to) the special characteristics or functions of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, etc. . (x) Vectors, host cells and recombination methods Recombinant methods can also be used to produce antibodies. To recombinantly produce anti-antigen antibodies, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further colonization (amplification of DNA) or performance. The DNA encoding the antibody can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of the antibody). A variety of carriers can be used. The vector components generally include (but are not limited to) one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer components, promoters, and transcription termination sequences. (a) Signal sequence components The antibody of the present invention can not only be directly recombinantly produced, but also can be produced in the form of a fusion polypeptide fused with a heterologous polypeptide. The heterologous polypeptide is preferably a signal sequence or a specific cleavage site at the N-terminus of the mature protein or polypeptide. Other peptides. The selected heterologous signal sequence is preferably a signal sequence that can be recognized and processed by the host cell (ie, cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced with, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leader sequences. For yeast secretion, the native signal sequence can be replaced with, for example, the yeast invertase leader sequence, factor leader sequence (including yeast (Saccharomyces ) And Kluyveromyces (Kluyveromyces )α-factor leader sequence) or acid phosphatase leader sequence, Candida albicans (C. albicans ) The glucoamylase leader sequence or the signal described in WO 90/13646. In mammalian cell performance, mammalian signal sequences and viral secretion leader sequences (such as herpes simplex gD signal) can be used. (b) Origin of replication Both expression and selection vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in a selection vector, this sequence is a sequence that enables the vector to replicate independently of the host chromosomal DNA, and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for use in various bacteria, yeasts and viruses. The origin of replication from plastid pBR322 is suitable for most Gram-negative bacteria, the 2μ plastid origin is suitable for yeast, and various viral origins (SV40, polyoma virus, adenovirus, VSV or BPV) are suitable For the selection of vectors in mammalian cells. Generally speaking, mammalian expression vectors do not require an origin of replication component (since the SV40 origin contains an early promoter, it is usually only possible to use this origin). (c) Select gene components Expression and selection vectors can contain selection genes, also known as selectable markers. Typical selection genes encode the following proteins: (a) confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate or tetracycline), ( b) Supplementing auxotrophic deficiency, or (c) Supplying key nutrients that cannot be obtained from complex media, such as supplying the gene encoding D-alanine racemase from Bacillus. One example of the selection process uses drugs to arrest the growth of host cells. Those cells that have been successfully transformed by the heterologous gene produce proteins that exhibit drug resistance and therefore survive the selection protocol. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells capable of receiving nucleic acid encoding antibodies, such as DHFR, glutamine synthase (GS), thymidine kinase, metal Thioprotein-I and metallothionein-II (preferably primate metallothionein gene), adenosine deaminase, ornithine decarboxylase, etc. For example, by culturing cells transformed with the DHFR gene in a medium containing methotrexate (Mtx) (a competitive antagonist of DHFR) to identify transformants. Under these conditions, the DHFR gene is amplified together with any other co-transformed nucleic acids. A Chinese hamster ovary (CHO) cell line lacking endogenous DHFR activity (such as ATCC CRL-9096) can be used. Alternatively, the cells transformed with the GS gene can be identified by culturing the transformants in a medium containing L-methionine sulfoximine (Msx) (an inhibitor of GS). Under these conditions, the GS gene is amplified together with any other cotransformed nucleic acids. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above. Alternatively, cells encoding the antibody of interest can be selected by growing cells in a medium containing a selection agent for a selectable marker (such as aminoglycoside antibiotics, such as kanamycin, neomycin, or G418) DNA sequence, wild-type DHFR gene and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) transformed or co-transformed host cells (especially wild-type hosts containing endogenous DHFR) . See U.S. Patent No. 4,965,199. The selection gene suitable for yeast is the one that exists in yeast plastid YRp7trp 1 gene (Stinchcomb et al.,Nature , 282:39 (1979)).trp The 1 gene provides a selection marker for mutant yeast strains that cannot grow in tryptophan (for example, ATCC No. 44076 or PEP4-1). Jones,Genetics , 85:12 (1977). Then the yeast host cell genometrp 1 The existence of dysfunction provides an effective environment for detecting transformation by cultivating in the presence of leukosan. Similarly,Leu 2 Defective yeast strains (ATCC 20,622 or 38,626) are carried byLeu 2 The known plastid supplement of genes. In addition, the vector derived from 1.6 μm circular plastid pKD1 can be used to transform Kluyveromyces. Alternatively, an expression system for large-scale production of recombinant bovine chymosin was reported for Kluyveromyces lactis. Van den Berg,Bio/Technology , 8:135 (1990). A stable multi-copy expression vector for secreting mature recombinant human serum albumin using Kluyveromyces industrial strains has also been disclosed. Fleer et al.,Bio/Technology , 9:968-975 (1991). (d) Promoter components Expression and selection vectors usually contain a promoter recognized by the host organism and operably linked to the nucleic acid encoding the antibody. Promoters suitable for prokaryotic hosts includepho A promoter, β-endoamidase and lactose promoter system, alkaline phosphatase promoter, tryptophan (trp) promoter system and hybrid promoters (such as tac promoter). However, other known bacterial promoters are also suitable. The promoter used in the bacterial system will also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody. Known promoter sequences used in eukaryotes. Virtually all eukaryotic genes have an AT-enriched region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream from the start of transcription of various genes is the CNCAAT region, where N can be any nucleotide. The 3'end of most eukaryotic genes is the AATAAA sequence, which can be a signal for adding a poly A tail to the 3'end of the coding sequence. All these sequences are suitable for insertion into eukaryotic expression vectors. Examples of promoter sequences suitable for yeast hosts include promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, Phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate dismutase, pyruvate kinase, triose phosphate isomerase, glucose phosphate isomerase and glucokinase. Other yeast promoters with inducible promoters whose transcription is controlled by growth conditions are used in the following promoter regions: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism , Metallothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the utilization of maltose and galactose. EP 73,657 further describes vectors and promoters suitable for yeast expression. Yeast enhancers are also suitable for use with yeast promoters. For example, the transcription of antibodies from vectors in mammalian host cells can be controlled by promoters obtained from: such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus , Cell megavirus, retrovirus, hepatitis B virus, simian virus 40 (SV40) virus genome; or heterologous mammalian promoter (such as actin promoter or immunoglobulin promoter); heat shock promoter The restriction condition is that these promoters are compatible with the host cell system. The early and late promoters of SV40 virus are conveniently obtained as SV40 restriction fragments that also contain the origin of replication of SV40 virus. Conveniently obtain the immediate early promoter of human cytomegalovirus as a HindIII E restriction fragment. The use of bovine papilloma virus as a vector to express DNA in a mammalian host is disclosed in US Patent No. 4,419,446. Modifications of this system are described in US Patent No. 4,601,978. The performance of human beta-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus is also described in Reyes et al.,Nature 297:598-601 (1982). Alternatively, the long terminal repeat of Rous Sarcoma Virus can be used as a promoter. (e) Strengthen sub-element components The transcription of the DNA encoding the antibody of the present invention by higher eukaryotes is usually increased by inserting enhancer sequences into the vector. A variety of enhancer sequences from mammalian genes (hemoglobin, elastase, albumin, α-fetoprotein and insulin) are known. However, we usually use enhancers from eukaryotic cell viruses. Examples include the SV40 enhancer (bp 100-270) behind the replication origin, the cytomegalovirus early promoter enhancer, the polyoma virus enhancer behind the replication origin, and the adenovirus enhancer. See also Yaniv for enhancing elements used to activate eukaryotic promoters,Nature 297:17-18 (1982). The enhancer can be spliced to the 5'or 3'position of the antibody coding sequence in the vector, but is preferably located at the 5'position of the promoter. (f) Transcription termination component Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences necessary to terminate transcription and stabilize mRNA. Such sequences are usually available from the 5'and sometimes 3'untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One suitable transcription termination component is the polyadenylation region of bovine growth hormone. See WO94/11026 and the expression vectors disclosed therein. (g) Selection and transformation of host cells Suitable host cells for the selection or expression of DNA in the vectors herein are prokaryotic, yeast or higher eukaryotic cells as described above. Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (Escherichia ) (E.g. Escherichia coli), enterobacteria (Enterobacter ), Erwinia (Erwinia ), Klebsiella (Klebsiella ), Proteus (Proteus ), Salmonella (Salmonella ) (E.g. Salmonella typhimurium (Salmonella typhimurium )), Serratia (Serratia ) (E.g. Serratia marcescens (Serratia marcescans )) and Shigella (Shigella ) And Bacillus (Bacilli ) (Such as Bacillus subtilis (B. subtilis ) And Bacillus licheniformis (B. licheniformis ) (E.g. Bacillus licheniformis 41P disclosed in DD 266,710 published on April 12, 1989)), Pseudomonas (Pseudomonas ) (Such as Pseudomonas aeruginosa (P. aeruginosa )) and Streptomyces (Streptomyces ). Although other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable, a preferred E. coli selection host is E. coli 294 (ATCC 31,446). These examples are illustrative and not restrictive. Full-length antibodies, antibody fusion proteins and antibody fragments can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, such as when the therapeutic antibody is combined with a cytotoxic agent (such as a toxin) that exhibits the efficacy of destroying tumor cells. Time. Full-length antibodies have a longer half-life in the circulation. Production in E. coli is faster and more cost-effective. For the performance of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237 (Carter et al.), U.S. Patent No. 5,789,199 (Joly et al.), and U.S. Patent No. 5,840,523 (Simmons et al.). Secret the best translation initiation region (TIR) and signal sequence. See also Charlton,Methods in Molecular Biology , Volume 248 (Edited by B. K. C. Lo, Humana Press, Totowa, N.J., 2003), p. 245-p. 254, describing the performance of antibody fragments in E. coli. After expression, the antibody in the soluble fraction can be separated from the E. coli cell paste and can be purified by, for example, a protein A or G column depending on the isotype. The final purification can be performed similarly to the method of purification of antibodies expressed in CHO cells, for example. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for selection or expression of antibody-encoding vectors. Saccharomyces cerevisiae is most commonly used in lower eukaryotic host microorganisms (Saccharomyces cerevisiae ) Or ordinary baker's yeast. However, many other genera, species, and virus strains are commonly available and suitable for use here, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe ); Kluyveromyces hosts, such as Kluyveromyces lactis, Kluyveromyces fragilis (K. fragilis ) (ATCC 12,424), Kluyveromyces bulgaricus (K. bulgaricus ) (ATCC 16,045), Kluyveromyces wickerii (K. wickeramii ) (ATCC 24,178), Kluyveromyces (K. waltii ) (ATCC 56,500), Kluyveromyces gonii (K. drosophilarum ) (ATCC 36,906), Kluyveromyces thermotolera (K. thermotolerans ) And Kluyveromyces Max (K. marxianus ); Yarrowia (yarrowia ) (EP 402,226); Methanol Yeast (Pichia pastoris ) (EP 183,070); Candida (Candida ); Trichoderma reesei (Trichoderma reesia ) (EP 244,234); Neurosporum crassa (Neurospora crassa ); Saccharomyces spp (Schwanniomyces ), such as Schwann Yeast (Schwanniomyces occidentalis ); and filamentous fungi, such as Rhodospira (Neurospora ), Penicillium (Penicillium ), Curvularia (Tolypocladium ) And Aspergillus (Aspergillus ) Host, such as Aspergillus nidulans (A. nidulans ) And Aspergillus niger (A. niger ). For reviews discussing the use of yeast and filamentous fungi in the manufacture of therapeutic proteins, see, for example, Gerngross,Nat. Biotech. 22:1409-1414 (2004). Certain fungal and yeast strains can be selected in which the glycosylation pathway has been "humanized", resulting in the production of antibodies with partial or full human glycosylation patterns. See, for example, Li et al.,Nat. Biotech. 24:210-215 (2006) (Describe the humanization of glycosylation pathways in methanoly yeast); and Gerngross et al., supra. Host cells suitable for expressing glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants have been identified, as well as those from the armywormSpodoptera frugiperda ) (Caterpillar), Aedes aegypti (Aedes aegypti ) (Mosquito), Aedes albopictus (Aedes albopictus ) (Mosquito), Drosophila melanogaster (Drosophila melanogaster ) (Drosophila) and Bombyx mori (Bombyx mori ) The corresponding licensed insect host cell of the host. Various virus strains used for transfection are publicly available, such as Spodoptera alfalfa (Autographa californica ) The L-1 variant of NPV and the Bm-5 virus strain of Bombyx mori NPV, and the viruses according to the present invention can be used as viruses herein, especially for transfecting Mythimna separata cells. Cotton, corn, potato, soybean, petunia, tomato, duckweed (Lemweed (Lemnaceae )), alfalfa (Medicago truncatula (M. truncatula )) and tobacco plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describe PLANTIBODIES for producing antibodies in monogenic plantsTM technology). Vertebrate cells can be used as hosts, and proliferation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are monkey kidney CV1 cell lines (COS-7, ATCC CRL 1651) transformed by SV40; human embryonic kidney cell lines (293 cells or sub-selected for growth in suspension culture) 293 cells, Graham et al.,J. Gen Virol. 36:59 36:59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL 10); Mouse Sertoli cells (TM4, Mather,Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA , ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver cancer cell line (Hep G2). Other suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp2/0. For reviews of certain mammalian host cell strains suitable for antibody production, see, for example, Yazaki and Wu,Methods in Molecular Biology , Volume 248 (Edited by B. K. C. Lo, Humana Press, Totowa, N.J., 2003), pages 255-268. Transform host cells with the above-mentioned expression or selection vectors for antibody production, and culture them in a conventional nutrient medium modified to induce promoters, select transformants, or amplify genes encoding the desired sequence . (h) Culturing host cells The host cells used to produce the antibodies of the present invention can be cultured in various media. Commercially available media, such as Han's F10 (Ham's F10, Sigma), minimum essential medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM) ) (Sigma), suitable for culturing host cells. In addition, any of the culture media described in the following documents can be used as the culture medium for these host cells: Ham et al.,Meth. Enz. 58:44 (1979); Barnes et al.,Anal. Biochem. 102:255 (1980); U.S. Patent No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 4,560,655; or No. 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re.30,985. Any of these media can optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), Nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN)TM Drugs), trace elements (defined as inorganic compounds, usually present in final concentrations in the micromolar range), and glucose or equivalent energy sources. It may also include any other necessary supplements at appropriate concentrations known to those skilled in the art. The culture conditions (such as temperature, pH, and the like) are the conditions used in the previously selected host cells for expression, and are obvious to those skilled in the art. (xi) Antibody purification When using recombinant technology, the antibody can be produced intracellularly, in the periplasmic space or directly secreted into the culture medium. If the antibody is produced in the cell, as the first step, for example, centrifugation or ultrafiltration is used to remove the particulate debris of the host cell or the dissolved fragment. Carter et al,Bio/Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. In short, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonamide (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. In the case of antibody secretion into the culture medium, a commercially available protein concentration filter (such as an Amicon or Millipore Pellicon ultrafiltration unit) is usually first used to concentrate the supernatant layer of these performance systems. Protease inhibitors such as PMSF can be included in any previous steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants. For example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography can be used to purify the antibody composition prepared from cells, and affinity chromatography is one of the generally preferred purification steps. The suitability of protein A as an affinity ligand depends on the species and the isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al.,J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al.,EMBO J. 5:15671575 (1986)). Although the matrix to which the affinity ligand is attached is most often agarose, other matrices can be used. Compared with the flow rate and processing time that can be achieved with agarose, mechanically stable matrices (such as controlled pore glass or poly(styrene divinyl) benzene) have a faster flow rate and shorter processing time. When the antibody contains CH In 3 domains, Bakerbond ABXTM Resin (J. T. Baker; Phillipsburg, N.J.) is suitable for purification. Depending on the antibody to be recovered, other techniques for protein purification, such as ion exchange column fractionation, ethanol deposition, reverse phase HPLC, silica chromatography, heparin SEPHAROSE™ chromatography, anion or cation exchange resins (such as Polyaspartic acid column) chromatography, chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation. Generally speaking, various methods for preparing antibodies for research, testing and clinical use are well established in this technology, consistent with the above methods and/or considered suitable for the specific antibody of interest by those familiar with this technology .C. Choose a biologically active antibody The antibodies produced as described above can be subjected to one or more "biological activity" analysis methods to select antibodies with beneficial properties from a therapeutic point of view or to select formulations and conditions that retain the biological activity of the antibodies. The ability of the antibody to bind to the elevated antigen can be tested. For example, for an anti-PDL1 antibody, the antigen binding properties of the antibody can be evaluated in an analytical method that detects the ability to bind to PDL1. In some embodiments, the binding of antibodies can be determined by saturation binding; ELISA; and/or competition analysis (such as RIA's). Similarly, other biological activity analyses can be performed on antibodies, for example to assess their effectiveness as therapeutic agents. Such analysis methods are known in the art and depend on the intended use of the target antigen and antibody. For example, the biological effects of PD-L1 blocked by antibodies can be found in CD8+ T cells, lymphocytic choriomeningitis virus (LCMV) mouse models and/or syngeneic tumor models (such as described in US Patent 8,217,149) In the assessment. In order to screen for antibodies that bind to a specific epitope on the antigen of interest (for example, the anti-PDL1 antibody in the blocking example binds to PD-L1), conventional cross-blocking analysis methods (such asAntibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Or, for example, Champe et al.J. Biol. Chem. 270:1388-1394 (1995) described epitope mapping to determine whether the antibody binds to the relevant epitope.D. Preparation of formulation After the relevant antibodies are prepared (for example, the techniques for making antibodies that can be formulated as disclosed herein will be described in detail below and are known in the art), a pharmaceutical formulation comprising them is prepared. In certain embodiments, the antibody to be formulated is not pre-lyophilized and the relevant formulation herein is an aqueous formulation. In certain embodiments, the antibody is a full-length antibody. In one embodiment, the antibody in the formulation is an antibody fragment, such as F(ab')2 , It may be necessary to solve the problem that the full-length antibody does not appear in this case (such as splicing the antibody into Fab). The therapeutically effective amount of the antibody present in the formulation is determined by considering, for example, the required dose volume and mode of administration. About 25 mg/mL to about 150 mg/mL, or about 30 mg/mL to about 140 mg/mL, or about 35 mg/mL to about 130 mg/mL, or about 40 mg/mL to about 120 mg/mL , Or about 50 mg/mL to about 130 mg/mL, or about 50 mg/mL to about 125 mg/mL, or about 50 mg/mL to about 120 mg/mL, or about 50 mg/mL to about 110 mg /mL, or about 50 mg/mL to about 100 mg/mL, or about 50 mg/mL to about 90 mg/mL, or about 50 mg/mL to about 80 mg/mL, or about 54 mg/mL to about 66 mg/mL is an exemplary antibody concentration in the formulation. An aqueous formulation of the antibody contained in a pH buffered solution is prepared. The buffer of the present invention has a pH in the range of about 5.0 to about 7.0. In certain embodiments, the pH is in the range of about 5.0 to about 6.5, the pH is in the range of about 5.0 to about 6.4, the pH is in the range of about 5.0 to about 6.3, the pH is in the range of about 5.0 to about 6.2, and the pH is about 5.0. To about 6.1, pH in the range of about 5.5 to about 6.1, pH in the range of about 5.0 to about 6.0, pH in the range of about 5.0 to about 5.9, pH in the range of about 5.0 to about 5.8, pH in the range of about 5.1 To about 6.0, pH in the range of about 5.2 to about 6.0, pH in the range of about 5.3 to about 6.0, pH in the range of about 5.4 to about 6.0, pH in the range of about 5.5 to about 6.0, pH in the range of about 5.6 To about 6.0, the pH is in the range of about 5.7 to about 6.0, or the pH is in the range of about 5.8 to about 6.0. In certain embodiments of the invention, the formulation has a pH of 6.0 or about 6.0. In certain embodiments of the invention, the formulation has a pH of 5.9 or about 5.9. In certain embodiments of the invention, the formulation has a pH of 5.8 or about 5.8. In certain embodiments of the invention, the formulation has a pH of 5.7 or about 5.7. In certain embodiments of the invention, the formulation has a pH of 5.6 or about 5.6. In certain embodiments of the invention, the formulation has a pH of 5.5 or about 5.5. In certain embodiments of the invention, the formulation has a pH of 5.4 or about 5.4. In certain embodiments of the invention, the formulation has a pH of 5.3 or about 5.3. In certain embodiments of the invention, the formulation has a pH of 5.2 or about 5.2. Examples of buffers that control the pH in this range include histidine (such as L-histidine) or sodium acetate. In some embodiments, the buffer contains histidine acetate or sodium acetate at a concentration of about 15 mM to about 25 mM. In certain embodiments of the present invention, the buffer contains a concentration of about 15 mM to about 25 mM, about 16 mM to about 25 mM, about 17 mM to about 25 mM, about 18 mM to about 25 mM, about 19 mM To about 25 mM, about 20 mM to about 25 mM, about 21 mM to about 25 mM, about 22 mM to about 25 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM or about 25 mM histidine acetate or sodium acetate. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.0. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.1. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.3. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.4. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.5. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.6. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.7. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.8. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.9. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 6.0. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 6.1. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 6.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH 6.3. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.3. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.4. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.5. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.6. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.7. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.8. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.9. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 6.0. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 6.1. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 6.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH 6.3. The formulation further includes sucrose in an amount of about 60 mM to about 240 mM. In some embodiments, the sucrose in the formulation is about 60 mM to about 230 mM, about 60 mM to about 220 mM, about 60 mM to about 210 mM, about 60 mM to about 200 mM, about 60 mM to about 190 mM. mM, about 60 mM to about 180 mM, about 60 mM to about 170 mM, about 60 mM to about 160 mM, about 60 mM to about 150 mM, about 60 mM to about 140 mM, about 80 mM to about 240 mM, About 90 mM to about 240 mM, about 100 mM to about 240 mM, about 110 mM to about 240 mM, about 120 mM to about 240 mM, about 130 mM to about 240 mM, about 140 mM to about 240 mM, about 150 mM to about 240 mM, about 160 mM to about 240 mM, about 170 mM to about 240 mM, about 180 mM to about 240 mM, about 190 mM to about 240 mM, about 200 mM to about 240 mM, about 80 mM to About 160 mM, about 100 mM to about 140 mM, or about 110 mM to about 130 mM. In some embodiments, the sucrose in the formulation is about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM. mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, or about 240 mM. In some embodiments, the antibody concentration in the formulation is about 40 mg/ml to about 125 mg/ml. In some embodiments, the antibody concentration in the formulation is about 40 mg/ml to about 120 mg/ml, about 40 mg/ml to about 110 mg/ml, about 40 mg/ml to about 100 mg/ml, about 40 mg/ml to about 90 mg/ml, about 40 mg/ml to about 80 mg/ml, about 40 mg/ml to about 70 mg/ml, about 50 mg/ml to about 120 mg/ml, about 60 mg /ml to about 120 mg/ml, about 70 mg/ml to about 120 mg/ml, about 80 mg/ml to about 120 mg/ml, about 90 mg/ml to about 120 mg/ml, or about 100 mg/ml ml to about 120 mg/ml. In some embodiments, the antibody concentration in the formulation is about 60 mg/ml. In some embodiments, the antibody concentration in the formulation is about 65 mg/ml. In some embodiments, the antibody concentration in the formulation is about 70 mg/ml. In some embodiments, the antibody concentration in the formulation is about 75 mg/ml. In some embodiments, the antibody concentration in the formulation is about 80 mg/ml. In some embodiments, the antibody concentration in the formulation is about 85 mg/ml. In some embodiments, the antibody concentration in the formulation is about 90 mg/ml. In some embodiments, the antibody concentration in the formulation is about 95 mg/ml. In some embodiments, the antibody concentration in the formulation is about 100 mg/ml. In some embodiments, the antibody concentration in the formulation is about 110 mg/ml. In some embodiments, the antibody concentration in the formulation is about 125 mg/ml. In some embodiments, a surfactant is added to the antibody formulation. Exemplary surfactants include nonionic surfactants, such as polysorbate (e.g., polysorbate 20, 80, etc.) or poloxamer (e.g., poloxamer 188, etc.). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of particles in the formulation and/or reduces adsorption. For example, the surfactant may be present in the formulation in an amount of about 0.001% to about 0.5% (w/v). In some embodiments, the surfactant (e.g., polysorbate 20) is about 0.005% to about 0.2%, about 0.005% to about 0.1%, about 0.005% to about 0.09%, about 0.005% to about 0.08%, About 0.005% to about 0.07%, about 0.005% to about 0.06%, about 0.005% to about 0.05%, about 0.005% to about 0.04%, about 0.008% to about 0.06%, about 0.01% to about 0.06%, about 0.02 % To about 0.06%, about 0.01% to about 0.05%, or about 0.02% to about 0.04%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.005% or about 0.005%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.006% or about 0.006%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.007% or about 0.007%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.008% or about 0.008%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.009% or about 0.009%. In certain embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount of about 0.01% or about 0.01%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.02% or about 0.02%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.03% or about 0.03%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.04% or about 0.04%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.05% or about 0.05%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.06% or about 0.06%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.07% or about 0.07%. In certain embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount of about 0.08% or about 0.08%. In certain embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount of about 0.1% or about 0.1%. In certain embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount of about 0.2% or about 0.2%. In certain embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount of about 0.3% or about 0.3%. In certain embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount of about 0.4% or about 0.4%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of about 0.5% or about 0.5%. In one embodiment, the formulation contains the reagents identified above (for example, antibodies, buffers, sucrose, and/or surfactants) and is substantially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, Chlorobutanol and benzethonium chloride. In another embodiment, a preservative may be included in the formulation, especially when the formulation is a multi-dose formulation. The concentration of the preservative may range from about 0.1% to about 2%, preferably from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers (such as those described in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)) may be included in the formulation, with limitations The condition is that it will not negatively affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration used and include: other buffers; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA ; Metal complexes (such as Zn-protein complexes); Biodegradable polymers, such as polyesters; and/or salt-forming counterions. The exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX® , Baxter International, Inc.). Certain exemplary sHASEGP and methods of use (including rHuPH20) are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase. The formulation herein may also contain more than one protein necessary for the specific indication to be treated, preferably a protein having complementary activities that do not adversely affect other proteins. For example, if the antibody is anti-PDL1, it can be combined with another agent, such as a chemotherapeutic agent and an anti-neoplastic agent. In some embodiments, the physical stability, chemical stability, or biological activity of the antibody in the formulation is evaluated or measured. It is known in the art that any of the methods described in the examples herein can be used to assess the stability and biological activity of the antibody in the formulation. For example, the stability of the antibody in the formulation can be measured by (but not limited to) the following measurements: size exclusion chromatography (SEC or SE-HPLC), imaging capillary isoelectric focusing (ICIEF), peptide localization, small Volume shading (HIAC) analysis and capillary electrophoresis (CE) techniques, such as CE-sodium dodecyl sulfate (CE-SDS) and CE-glycan analysis. In some embodiments, the antibody in the formulation is stable at -20°C for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months. Months, at least about 18 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 3 years, or at least about 4 years. In some embodiments, the antibody in the formulation is stable at 2°C to 8°C (eg, 5°C) for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months. In some embodiments, the stability of the antibody (ie, antibody monomer) in the formulation is measured by size exclusion chromatography after storage. In some embodiments, the stability of the antibody (ie, antibody monomer) in the formulation is measured by imaging capillary isoelectric focusing after storage. In some embodiments, after storage at -20°C for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months, the antibody monomer in the formulation is compared to the total protein (eg Including antibodies and aggregates) the percentage is greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%. In some embodiments, after storage at 2°C to 8°C (eg 5°C) for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months, the antibody monomer in the formulation Compared to (e.g., including antibodies and aggregates), the percentage is greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, About 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%. In some embodiments, stirring is performed at room temperature (for example, about 15°C to 25°C) for at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, After at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, or at least about 24 hours, the antibody monomer in the formulation is compared to (e.g., includes antibody And aggregates) percentage is greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90% , About 91%, about 92%, about 93%, about 94%, or about 95%. In some embodiments, after storage at -20°C for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months, all aggregates (such as high molecular weight substances and low The percentage of molecular weight substances) is less than about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, Any of about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, after storage at 2°C to 8°C (eg 5°C) for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months, all aggregates in the formulation (E.g. high molecular weight substances and low molecular weight substances) are less than about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about Any of 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, stirring at room temperature (eg, about 15°C to 25°C) for at least about 2 months, at least about 4 months, at least about 6 months, at least about 8 months, at least about 10 months, After at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, or at least about 24 months, all aggregates (such as high molecular weight substances and low The percentage of molecular weight substances) is less than about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, Any of about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In any of the embodiments herein, the stable formulation may be stored in a glass bottle, metal alloy container, or intravenous (IV) bag. In some embodiments, the metal alloy is 316L stainless steel or Hester alloy. The formulation used for in vivo administration should be sterile. This is easily achieved by filtration through a sterile filter membrane before or after preparing the formulation.III. Methods of treatment and administration of antibody formulations According to known methods (such as intravenous administration (for example, in a bolus or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, The formulation is administered to mammals (preferably humans) in need of antibody treatment via oral, topical or inhalation routes. In one embodiment, the formulation is administered to the mammal by intravenous administration. For such purposes, the formulation can be injected, for example, using a syringe or via an IV catheter. In one embodiment, the formulation is administered to the mammal by subcutaneous administration. The appropriate dose of the antibody ("therapeutically effective amount") will depend on the following, such as the condition to be treated, the severity and progression of the condition, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, and the patient's clinical history And the response to the antibody, the type of antibody used and the judgment of the attending physician. The antibody is appropriately administered to the patient at one time or over a series of treatments, and can be administered to the patient at any time from the date of diagnosis. The antibody can be administered as a single treatment or in combination with other drugs or therapies suitable for the treatment of related conditions. As a general recommendation, the therapeutically effective amount of antibody administered to humans by one or more administrations will be in the range of about 0.01 to about 50 mg/kg of the patient's body weight. In some embodiments, the antibody used is, for example, about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg /kg. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosage regimens are also applicable. In one embodiment, the anti-PDL1 antibody described herein is administered at about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, A dose of about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg is administered to humans. The dose can be administered in a single dose or multiple doses (e.g., 2 or 3 doses), such as infusion. The dose of antibody administered in combination therapy may be lower than that of single therapy. The progress of this therapy is easily monitored by conventional techniques. The formulations containing the anti-PDL1 antibodies described herein can be used in a variety of in vitro and in vivo diagnostic and therapeutic applications. For example, the antibody-containing formulation can be administered to an individual to treat a disease or disorder (for example, a disease or disorder mediated by the interaction of PD-1 and PD-L1) In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is locally advanced cancer or metastatic cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological cancers, bladder cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, Lung cancer (e.g. non-small cell lung cancer), lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, saliva cancer, gastric cancer, thymic epithelial cell carcinoma, thyroid cancer, and head and neck Squamous cell carcinoma. In some embodiments, the individual being treated has PD-L1 positive cancer cells (eg, detected by IHC). In some embodiments, the disease or condition is an infection. In some embodiments, the infection is a persistent infection. In some embodiments, the infection is a viral, bacterial, fungal, worm, or protozoan infection. In some embodiments, the viral infection is selected from the group consisting of cytomegalovirus, Epstein-Barr virus, hepatitis B, hepatitis C virus, herpes virus, measles virus, influenza, Human immunodeficiency virus, human T cell virus, lymphocytic choroid meningitis virus, respiratory syncytial virus and/or rhinovirus. In some embodiments, the bacterial infection is selected from the group consisting of: Helicobacter (Helicobacter spp. ), Mycobacterium (Mycobacterium spp. ), Porphyromonas (Porphyromonas spp. ), Chlamydia (Chlamydia spp. ), Salmonella (Salmonella spp. ), Listeria (Listeria spp. ), Streptococcus (Streptococcus spp. ), Haemophilus (Haemophilus spp. ), Neisseria (Neisseria spp. ), Klebsiella (Klebsiella spp. ), Borrelia (Borrelia spp. ), Bacteroides (Bacterioides spp. ) And Treponema (Treponema spp. ). In some embodiments, the protozoan infection is selected from the group consisting of: Leishmania (Leishmania spp. ), Plasmodium falciparum (Plasmodium falciparum ), live Schistosoma (Schistosoma spp. ), Toxoplasma (Toxoplasma spp. ), Trypanosoma (Trypanosoma spp. ) And tapeworm (Taenia spp. ). In some embodiments, the fungal infection is selected from the group consisting of: blastomycosis, coccidioidomycosis (coccidioiodmycosis ), histoplasmosis (histoplamsosis ), candidiasis (candidiasis ), cryptococcosis (cryptococcosis ), Aspergillosis (aspergillossi ), mucormycosis (mucomycosis ) And pneumocysticercosis (pneumocystosis ). In some embodiments, the disease or condition is an inflammatory disease. In some embodiments, the inflammatory disease is selected from the group consisting of acute disseminated encephalomyelitis, Addison's disease, Alzheimer's disease, ankylosing spondylitis, Antiphospholipid antibody syndrome, atherosclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, Behcet's disease, Berger's disease, bullous pemphigoid , Celiac disease, Chagas' disease, cholangitis, Crohn's disease, dermatomyositis, type 1 diabetes, spontaneous nephritis, Goodpasture's syndrome, transplant Material-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, urticaria, super-IgE syndrome, idiopathic thrombocytopenia Purpura, lupus erythematosus, lupus nephritis, multiple sclerosis, myasthenia gravis, organ transplant rejection, Parkinson's disease, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, psoriasis , Raynaud's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, temporal arteritis, thyroiditis, ulcerative colitis, uveitis, vasculitis and Wei Wegener's granulomatosis. In some embodiments, the antibody-containing formulation can be combined with another therapeutic agent and administered to an individual to treat a disease or condition. For example, to treat cancer, the anti-PDL1 antibody formulations described herein can be administered in combination with another anti-cancer therapy (e.g., chemotherapy or a different antibody therapy).IV. Product or set In another embodiment of the present invention, an article or kit containing a container is provided, and the container contains the aqueous pharmaceutical formulation of the present invention; and instructions for use thereof are provided as appropriate. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed of a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or Hester alloy). An exemplary container is a 300 cc metal alloy container (for example for storage at -20°C). Another exemplary container may be a 10-50 cc glass bottle (for example for storage at 2-8°C). For example, the container can be a 10 cc, 15 cc, 20 cc, or 50 cc glass bottle. The container contains the formulation and the label on the container or the label associated with the container may indicate instructions for use. The product may further include other materials that are desirable from a commercial and user point of view, including other buffers, diluents, filter paper, needles, syringes, and package inserts with instructions for use. In some embodiments, the preparation further includes one or more other agents (eg, chemotherapeutic agents and anti-neoplastic agents). Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes. It is considered that the description is sufficient to enable those skilled in the art to implement the present invention. Those skilled in the art will be aware of various modifications of the present invention other than those shown and described herein from the above description, and these modifications are within the scope of the appended application. All publications, patents and patent applications cited in this article are all incorporated herein by reference for all purposes. Instance Refer to the following examples to fully understand the present invention. However, it should not be regarded as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art can make various modifications or changes to them and they are included in the spirit and scope of this application and the scope of the accompanying patents.Instance 1 :anti PDL1 Development of antibody formulations Anti-PDL1 antibody (α-PDL1) is a deglycosylated IgG1 antibody derived from CHO, which is intended to restore T cell function by inhibiting the PDL1/PD1 interaction. Attacks at the beginning of development include potential Trp oxidation and glycosylation in or near the CDR region and some methionine oxidation. Previous studies on robustness indicated that a higher pH than the previous target (pH 5.5) is optimal. The target dose is a fixed dose, but also covers weight-based doses. Perform analytical studies to analyze the stability of multiple formulations and select one formulation (60 mg/mL α-PDL1, 20 mM His AcO pH 5.8, 120 mM sucrose, 0.04% PS20). The initial formulation research supports up to three years of stability of the bulk drug (DS) and drug product (DS).Methods and materials manufacture α-PDL1 Formulation Research on formulations of α-PDL1 materials undergoing ultrafiltration/diafiltration effects. A 10,000 Dalton dialysis cassette was used to dialyze the material into a variety of formulation buffers. After dialysis, adjust the protein concentration to reach the target concentration and add 10% PS20 stock solution to reach the target PS20 concentration. The compounding material is aseptically filled into a 2 cc Forma Vitrum glass bottle with a filling volume of 1 mL and sealed with a 13 mm Daikyo 777-1 stopper. The samples are stored upright at 5°C, 25°C, or 40°C.Color, appearance and transparency (CAC) At room temperature, using a black and white background, visually determine the color, appearance and transparency of the sample under white fluorescent light, such as the European Pharmacopoeia (EP) method (Council of Europe.European Pharmacopoeia , 2008, 7th edition, EP 2.2.2 and EP 2.2.1). Fill a 3cc glass bottle with 1 mL of each test sample. Use a negative control (pure water) with the corresponding sample volume for comparison.Protein concentration measurement The sample volume was diluted to about 0.5 mg/mL with 0.9% saline, and the protein concentration was determined by UV absorbance measurement on an Agilent 8453 spectrophotometer (Santa Clara, CA.). The sample is blank for 0.9% normal saline and the A at about 280 nmmax And also measure the absorbance at 320 nm. Calculation Amax With A320 The difference between gets corrected Amax , Use it to use 1.5mL cm-1 mg-1 The absorbency determines the final protein concentration.Turbidity measurement On the Agilent 8453 spectrophotometer, using a 1-cm path length, measure the average optical density of the sample at 350 nm in a quartz cuvette. Use pure water as a blank.Shading method for microscopically visible particles (HIAC Analysis ) Count the sample particles by measuring the shading by Model 9703 HIAC-Royco (HACH, Loveland, CO.). Use PharmSpec v2.0 to list the average cumulative number of particles per milliliter of each sample ≥ 2 mm, ≥ 5 mm, ≥ 10 mm, and ≥ 25 mm. Each test takes four readings that consume a total of 1.6 mL of each sample. The first reading is discarded, and the remaining 3 readings are averaged.Size exclusion chromatography (SEC or SE-HPLC) The size variant distribution was determined by size exclusion chromatography (SEC) using TosoHaas Bioscience column G3000 SWXL (South San Francisco, CA.) at 30°C on an Agilent 1200 HPLC (Santa Clara, CA., USA). All samples were injected undiluted with 50 μg onto the column and dissolved in 60 minutes, with UV absorption at 280 nm. Two different SEC methods were used for sample testing. Method 1 uses 0.20 M potassium phosphate, 0.25 M potassium chloride, pH 6.2, and method 2 uses 0.20 M potassium phosphate, 0.25 M potassium chloride, pH 6.2, and uses 10 vol% isopropanol as the mobile phase. The results are reported as a relative percentage of the peak area of the total area under the curve.Imaging capillary isoelectric focusing (ICIEF) The iCE280 analyzer (ProteinSimple) with a fluorocarbon-coated capillary filter element (100 µm × 5 cm) was used to evaluate the charge variant distribution by iCIEF. Ampholyte solution consists of 0.35% methyl cellulose (MC), 0.75% Pharmalyte 3-10 carrier ampholyte, 4.2% Pharmalyte 8-10.5 carrier ampholyte, 0.2% pI labeled 7.40 and 0.15% pI labeled 9.77 in pure water Composition in the mixture. The anolyte is 80 mM phosphoric acid and the catholyte is 100 mM sodium hydroxide, both of which are contained in 0.10% methylcellulose. The samples were diluted in pure water and CpB was added to each diluted sample at an enzyme ratio of 1:100, followed by incubation at 37°C for 20 minutes. The CpB-treated sample was mixed with the amphoteric electrolyte solution, and then focused by introducing a 1500 V potential for 1 minute, and then a 3000 V potential for 10 minutes. By passing 280 nm ultraviolet light through the capillary tube and entering the lens of the charge-coupled device digital camera, an image of the focused α-PDL1 charge variant is obtained. This image is then analyzed to determine the distribution of various charge variants.Peptide mapping Use peptide localization technology to monitor the oxidation of tryptophan (W) and methionine (M). In order to generate α-PDL1 peptide localization, after exposing the protein to dithiothreitol (DTT) and iodoacetic acid (IAA), the pancreas is used to reduce the disulfide bond and change the resulting free thiol to produce carboxymethyl derivatives. Proteases digest proteins. The resulting peptides were separated by reverse phase high performance liquid chromatography (RP-HPLC) and monitored at 214 nm. The mass of tryptic peptide was determined by LC-MS analysis of the separated digestion mixture using ThermoFisher Scientific LTQ-Orbitrap mass spectrometer.result Choosing a buffer system During formulation development, two buffer systems were evaluated. One is 20 mM histidine acetate with 240 mM sucrose, pH 5.5, and the other is 200 mM arginine succinate, pH 5.5. Accelerated stability studies revealed that α-PDL1 has better stability in histidine acetate buffer than in succinate arginine buffer (Table 1). Therefore, histidine acetate was selected for further development of the formulation.table 1. The zero-order decomposition rate of α-PDL1 of the main peak of ICIEF and SE-HPLC in histidine acetate and arginine succinate buffer at 30℃
Figure 108108366-A0304-0001
 Note: All formulations are stored at 30°C for up to 1 month. Analyzed by ICIEF and SE-HPLC; * 150 mg/mL containing α-PDL1 20 mM L-histidine acetate, 240 mM sucrose and 0.02% (w/v) polysorbate 20, pH 5.5; * * 150 mg/mL 200 mM arginine succinate containing α-PDL1, 0.02% (w/v) polysorbate 20, pH 5.5.Choose a stabilizer Based on the ability of sucrose to protect proteins from freeze/thaw-induced aggregation and to be used as a cryoprotectant during long-term frozen storage of the drug substance (DS) and subsequent storage of the drug product (DP) at 2°C to 8°C, sucrose (120 mM ) As a stabilizer for α-PDL1 liquid formulations. During the development of the formulation, 20 mM L-histidine acetate, pH 5.5, 0.02% (w/v) polysorbate 20, and 50 mg/mL α in various concentrations of sucrose in the range of 0 mM to 120 mM -PDL1 performs five freeze/thaw cycles. The product quality measured by SE-HPLC indicated that 60 mM sucrose was sufficient to prevent freezing/thawing-induced increase in α-PDL1 HMWS (Table 2). In addition, 120 mM sucrose was shown to maintain the stability of the bulk drug when stored frozen at -20°C for at least 6 months (Table 3). Therefore, based on the results of freezing/thawing studies and the long-term stability of the drug substance when stored at -20°C, 120 mM sucrose was selected as the cryoprotectant for the liquid formulation of α-PDL1.table 2. The effect of sucrose concentration on the stability of α-PDL1 SE-HPLC percentage high molecular weight substances during freezing and thawing
Figure 108108366-A0304-0002
 Note: All formulations contain 50 mg/mL α-PDL1, 20 mM L-histidine acetate, 0.02% (w/v) polysorbate 20, pH 5.5. The following are used for analysis: SE-HPLC; F/T = freeze/thaw; HMWS = high molecular weight substance; SY = slightly yellow; CL = clear; PFVP = practically no visible particles.table 3. Long-term stability data of R&D batch of α-PDL1 API
Figure 108108366-A0304-0003
 Note: All formulations contain 60 mg/mL 20 mM L-histidine acetate containing α-PDL1, 120 mM sucrose, 0.04% PS20, and pH 5.8. This study uses 25cc 316L stainless steel cans; NA=not applicable; CAC=color, appearance and transparency; SY=slight yellow, CL=clear, PFVP=actually no visible particles; HMW=high molecular weight; LMW=low molecular weight; ICIEF = imaging capillary isoelectric focusing; CE-SDS = sodium dodecyl sulfate capillary electrophoresis; NT = not tested; TBD = to be determined.Pre-allocation robustness study: select protein concentration, pH Polysorbate 20 concentration Partial factorial design of experiment (DOE) was used to further examine the effect of α-PDL1 formulation parameters on protein stability. A total of 12 different α-PDL1 formulations were tested (10 experiments and 2 center points). The three factors that changed in the study were the pH range of 5.0-6.0 with 0.5 unit intervals, the protein concentration range of 40-120 mg/mL, and the polysorbate 20 concentration range of 0.005%-0.06% (w/v) (Table 4). All formulations were buffered with 20 mM histidine acetate with 120 mM sucrose, except for the last two formulations indicated in Table 4. The 25 mM histidine acetate formulation was evaluated because it was considered the worst case scenario in terms of oxidation risk. Evaluate 20 mM sodium acetate buffer as a backup buffer system and compare with histidine acetate buffer. The formulation was stored at 25°C for 2 months and 40°C for 1 month. Use JMP software (JMP, 9th edition, SAS Institute Inc., Cary, NC) to perform statistical analysis of the interaction between the formulation parameters on the stability data studied above.table 4. Α-PDL1 APIs and drug formulations evaluated in the DOE study
Figure 108108366-A0304-0004
 Note: Yesa Center pointb Worst case scenario: low protein concentration, high PS20 concentration, high histidine concentration;c 20 mM sodium acetate (Na-Ace) buffer for testing. Compared with pH 5.0 and 5.5, as measured by ICIEF at 40°C and 25°C, the main peak loss rate of the formulation at pH 6.0 is slightly lower (Figure 1A-Figure 1B and Figure 2A-Figure 2B, respectively). ICIEF observed that the concentration had no significant effect on the loss of the main peak. The analysis of formulation F1 showed that in ICIEF, the increase in acidic variants mainly caused the loss of the main peak, while the peak loss caused by the basic charge variants was not significant. Under the same storage conditions, as measured by SE-HPLC at 40°C and 25°C, the formulation with pH 6.0 also has a slower monomer peak loss rate (Figure 3A-Figure 3B and Figure 4A-Figure 4B, respectively) ). The analysis of formulation F1 showed that in SEC, at high temperature (ie 40°C and 25°C), both HMWS and LMWS formation caused monomer loss. Both SEC and ICIEF pH rate profiles revealed that pH 5.5-6.0 is the optimal pH range for α-PDL1. In order to achieve the best protein stability higher than pH 5.5 and allow the range of ±0.3 pH units in the formulated raw materials and drugs, the target of pH 5.8 is selected. The above formulation study also revealed that the 120 mg/mL α-PDL1 formulation in the range of 5.0-6.0, as determined by SE-HPLC, is compared to the 40 mg/mL formulation at the same pH due to higher HMWS formation The compound has a slightly higher but not significant monomer peak loss rate (Figure 3A-Figure 3B and Figure 4A-Figure 4B). Based on these data and in order to support formulations with improved product stability and to facilitate patient administration, a concentration of 60 mg/mL α-PDL1 was selected. As indicated by the above statistical analysis, it was observed that polysorbate 20 (PS20) in the concentration range of 0.005%-0.06% (w/v) had no effect on protein stability (Figure 1-Figure 4). It is known that the hydrogen peroxide impurities contained in the polysorbate 20 raw material can cause the oxidation of tryptophan (W) and methionine (M). L-histidine can also increase the above-mentioned oxidation risk. The samples of the worst-case scenario selected formulations containing higher concentrations of polysorbate 20 and L-histidine were analyzed by peptide mapping. The analysis results show that even the combination of higher histidine concentration (25mM histidine acetate buffer) and higher amount of PS20 (0.06% PS20) does not exhibit a significant oxidation risk (Table 5) and histidine buffer is suitable for Prepare α-PDL1.table 5. Percentage of Trp and M in the selected formulation by peptide positioning253 Oxidation
Figure 108108366-A0304-0005
 In order to evaluate the possible decomposition of PS20 in the formulations during storage, formulations F1 to F10 (Table 4) were stored at 40°C for 1 month. Store at 25℃ for 2 months, at 5℃ for 2 months or at 5℃ for 6 months. No decomposition of PS20 was observed in the evaluated formulations at any elevated temperature (ie 40°C and 25°C) and 5°C storage temperature. Changing the filling volume of the selected formulations (ie F1, F2, F3, and F6) to 7 ml (high filling) or 4 ml (low filling), and then storing at 5°C for 6 months will not affect the PS20 The decomposition rate has a significant effect (Figure 5). The formation of microscopically visible particles (SbVP) in different formulations stored at 5°C for 6 months was evaluated by HIAC analysis as a measure of stability (Table 6). No measurable changes in SbVP were observed in the tested formulations.table 6. HIAC data of SbVP formation after 6 months storage at 5℃
Figure 108108366-A0304-0006
 Note: Two 1 mL filled vials are combined for small volume HIAC analysis. Use freeze-thaw experiments to further study the stability of the formulation. Formulations F1 to F10 (Table 4) were stored at -20°C for five freeze-thaw cycles or stored at a high storage temperature of 5°C for 0 to 6 months and then analyzed by SEC and ICIEF for α-PDL1 monomer (Figure 6A and Figure 6B) and the main peak percentage in the formulation (Figure 6C and Figure 6D). After the freeze-thaw cycle and storage at the specified time point, no significant changes were observed in the monomer percentage and the main peak percentage. Five freeze-thaw cycles were performed during storage in a small stainless steel tank at -20°C for up to 6 months, followed by stability measurement by CAC, SEC and ICIEF (Table 7) to evaluate the raw materials in the F2 formulation Drug stability (Table 4). No change was observed after 6 months storage at -20°C.table 7. Stability of API in small stainless steel tanks stored at -20℃
Figure 108108366-A0304-0007
 Note: F/T = freeze/thaw; SY = slightly yellow; CL = clear. By performing three freezing and thawing cycles, and then storing in a stainless steel canister or a Hester alloy canister at -20°C, 5°C or 25°C for up to 3 months, then stability measurement by SEC to assess the content The stability of the drug substance in the formulation of 100 mg/mL α-PDL1, 20 mM histidine acetate, 120 mM sucrose, 0.04% PS20, pH 5.6 (Figure 7A and Figure 7B). At pH 5.6, no difference was observed in storage in small stainless steel tanks and small Hester alloy tanks. At -20°C, the bulk drug is stable for up to 3 months after three freeze-thaw cycles. Although there are slight differences between stainless steel canisters and Hester alloy canisters, both are suitable for bulk drug storage. Store up to 3 months in a 16 mL filled form in a 20cc vial at -5°C, 25°C or 40°C, and then conduct stability measurements by SEC and ICIEF, and assess that it contains 50 mg/mL α-PDL1, 20 Drug stability in the formulation of mM histidine acetate, 120 mM sucrose, 0.04% PS20, pH 5.6 (Figure 8A and Figure 8B). After three months of storage at 5°C, no changes were observed. At 40℃, the monthly decomposition rate of pH 5.6 by SEC and ICIEF analysis is 0.66% and 22%, respectively. The evaluation indicator of the buffer in the F12 formulation is based on the main peak decomposition rate measured by SE-HPLC and ICIEF (Table 8). Sodium acetate buffer provides protein stability similar to histidine acetate buffer. The two test formulations are 50 mg/mL containing α-PDL1, 20 mM L-histidine acetate, 120 mM sucrose and 0.04% (w/v) polysorbate 20, pH 5.5 and 0 mg/mL containing α-PDL1 is 20 mM sodium acetate, 120 mM sucrose and 0.04% (w/v) polysorbate 20, pH 5.5.table 8. The zero-order decomposition rate of α-PDL1 of the main peak of ICIEF and SE-HPLC in histidine acetate and sodium acetate buffer at 40℃
Figure 108108366-A0304-0008
 Note: All formulations are stored at 40°C for up to 1 month. Overall, the DoE design stability study revealed that at 40°C, ICIEF did not observe a significant effect of concentration on the loss of the main peak, while lower pH had a slightly faster loss of the main peak rate (Figure 1A-Figure 1B). At 40°C, SE-HPLC also did not observe significant interactions, however, higher concentration formulations showed faster monomer loss (Figure 3A-Figure 3B). It was also found that lower pH has faster monomer rate loss. Similar results were observed at 25°C (Figure 2A-2B and Figure 4A-4B). Statistical analysis reveals that there is no meaningful interaction (connection) between any test formulation parameters.Stirring and thermal stress research The stability of the drug under the stress of stirring in a glass bottle in the presence of increased concentration of PS20 was studied. In a 2cc glass bottle with various concentrations of PS20 in the range of 0.005% to 0.06%, in a 1 mL filling, a formulation containing 20 mM histidine acetate, 120 mM sucrose, and 57 mg/mL in pH 5.5 was evaluated. Then, before measuring the stability by SEC (Figure 9A) and measuring the turbidity (Figure 9B), the glass bottle was stirred at 70 rpm at room temperature for 3 days. The stability of formulations with a PS20 content between 0.005-0.06% during stirring remains unchanged. However, formulations lacking PS20 showed increased monomer loss due to increased HMWS. In this experiment, 0.005% PS20 is sufficient to protect the protein from the stirring stress in the glass bottle. Study the stability of the drug formulation (Table 4) when stored at various temperatures and times, and then subjected to the stirring stress in the glass bottle. Each formulation F1-F10 was evaluated in a 1 mL filling in a 2cc glass bottle. The glass bottle was stirred at room temperature at 70 rpm for 1 day, and then the stability was measured by SEC (Figure 10). In this experiment, stirring has no effect on the stability of medicines stored for a long time at 40°C, 25°C or 5°C. In order to support the IV bag transportation that usually occurs in the hospital environment, α-formulated in 20 mM histidine acetate, 240 mM sucrose, pH 5.5 with 0.005%-0.02% (w/v) polysorbate 20 is used. PDL1 conducts IV bag mixing research. By injecting 400-600 mg of α-PDL1 solution and using an orbital shaker at 100 rpm and stirring at 5°C for up to 6 hours, the most commonly used 250mL polyvinyl chloride containing isotonic sodium chloride solution (0.9% NaCl) (PVC) or polyolefin (PO) IV bags are evaluated. The results of the study support weight-based dosing and indicate that a minimum of 0.015% (w/v) polysorbate 20 is required in the protein solution to prevent the formation of visible particles (related to protein precipitation) during transportation (Table 9). In addition, in order to reduce the risk of decomposition of polysorbate 20 during storage, the concentration of polysorbate 20 was increased from 0.02% (w/v) to 0.04% (w/v).table 9. Study on mixing IV bags with different amounts of α-PDL1 drugs containing PS20
Figure 108108366-A0304-0009
 Note: 50 mg/mL of all formulations contain α-PDL1, 20 mM L-histidine acetate, 240 mM sucrose, pH 5.5. SE-HPLC was used for analysis. NT = not tested; CAC = color, appearance and clarity; CO = colorless; CL = clear; PFVP = practically no visible particles.α-PDL1 Stability assessment of formulations For the master cell bank (Master Cell Bank) and the working cell bank (Working Cell Bank) in the pH range of 5.2 to 6.3 in a formulation containing 20 mM histidine acetate, 120 mM sucrose and 0.04% PS20 The material was subjected to additional pH screening (Table 10). SE-HPLC and ICIEF analysis showed that pH 5.7-6.3 is chemically and physically quite stable and the allowable range of pH 5.5-6.3 in the formulation is appropriate (Figure 11A and Figure 11B). Higher pH decreases the decomposition rate of monomer and main peak, and the rate becomes flat between about pH 5.7 to 6.3.table 10. PH screening of formulations
Figure 108108366-A0304-0010
 To study the effect of excipients on the oxidation of tryptophan (W) and methionine (M) in α-PDL1 formulations. Peptide localization showed no significant increase in oxidation. A formulation containing 20 mM histidine acetate, 120 mM sucrose, 0.04% PS20 and a solution pH of 5.8 showed that when the formulation was stored at a high temperature for one month, there was no obvious oxidation of tryptophan and methionine Improve (Table 11).table 11. Trp, M in the selected formulation determined by peptide localization253 And M429 Percentage of oxidation
Figure 108108366-A0304-0011
 Note: All formulations of α-PDL1 contain 20 mM L-histidine acetate, 120 mM sucrose, 0.04% PS20, and pH 5.8. Based on the results of research and statistical analysis of these formulations, a liquid formulation composed of 60 mg/mL α-PDL1 containing 20 mM histidine acetate, 120 mM sucrose, 0.04% polysorbate 20, and a target pH of 5.8 was selected. Clinical research. A uniform dose of 1200 mg α-PDL1 per patient will be used for clinical trials. Choose a vial configuration with a nominal 20 mL filling (1200mg α-PDL1) in a 20 cc glass bottle to meet the target product profile. The freezing/thawing study was performed using the expected formulation containing 60 mg/mL α-PDL1 in 20 mM L-histidine acetate, 120 mM sucrose and 0.02% (w/v) polysorbate 20, pH 5.8. The analysis results after five freeze/thaw cycles confirmed that 120 mM sucrose protects α-PDL1 from freeze/thaw-induced aggregation (Table 12). The similar long-term stability of the liquid formulation is expected to indicate that it is stable for more than 6 months at 2-8°C (Table 13). This formulation is under continuous monitoring for 36 months. Table 14 shows the target formulation and test research scope of the raw material drug of the α-PDL1 API.table 12. Representative freeze/thaw stability data of R&D batches of α-PDL1 API
Figure 108108366-A0304-0012
 Note: Batch PP400L-02142013 contains 60 mg/mL 20 mM L-histidine acetate containing α-PDL1, 120 mM sucrose and 0.04% (w/v) polysorbate 20, pH 5.8. CL = clarification; SY = slightly yellow; PFVP = practically no visible particles; NA = not applicable, ICIEF = imaging capillary isoelectric focusing; CE-SDS = sodium dodecyl sulfate capillary electrophoresis; HMW = high molecular weight; LMW = Low molecular weight.table 13. Stability data of α-PDL1 drug development batch
Figure 108108366-A0304-0013
 Batch PP400L-02142013-DP contains 60 mg/mL 20 mM L-histidine acetate containing α-PDL1, 120 mM sucrose and 0.04% (w/v) polysorbate 20, pH 5.8. NA = not applicable; CAC = color, appearance and clarity; SY = yellowish, CL = clear, PFVP = practically free of visible particles; HMW = high molecular weight; LMW = low molecular weight; ICIEF = imaging capillary isoelectric focusing; CE-SDS = sodium dodecyl sulfate capillary electrophoresis, NT = not tested.table 14. Alpha-PDL1 API and drug target formulations and test research scope
Figure 108108366-A0304-0014
 Because α-PDL1 medicine (60 mg/mL) will be diluted in isotonic sodium chloride solution (0.9% NaCl) and then administered by infusion, the compatibility of the active ingredient was tested under the following simulated preparation and administration conditions And stability: 1) Dilute α-PDL1 in an infusion bag containing 0.9% NaCl in the range of 2.4-9.6 mg/ml (nominal concentration after dilution) to cover the dose range in clinical studies; 2) Short-term exposure In an infusion bag containing isotonic sodium chloride solution (the product contact surface material of the bag is made of PVC or polyolefin); 3) Use IV infusion line (the product contact surface is PVC or polyolefin); and 4) In the online filter Use 0.2 μm (filter membrane is PES). Store the samples at 2°C-8°C for 24 hours or expose them to scattered light at 30°C for 24 hours to test the samples. Use appropriate stability indicator methods to test the samples, including: testing purity by SE-HPLC and ICIEF, protein concentration (by UV), testing microscopic visible particles by opacity, color, transparency/opalescence and pH ( Table 15).table 15. Stability of α-PDL1 in 0.9% NaCl infusion bags with and without 0.2 μm in-line filter after dilution and storage at 5℃ or 30℃ for 24 hours
Figure 108108366-A0304-0015
 table 15 ( Continued ) : Stability of α-PDL1 in 0.9% NaCl infusion bags with and without 0.2 μm in-line filter after dilution and storage at 5℃ or 30℃ for 24 hours
Figure 108108366-A0304-0016
 table 15 ( Continued ) : Stability of α-PDL1 in 0.9% NaCl infusion bags with and without 0.2 μm in-line filter after dilution and storage at 5℃ or 30℃ for 24 hours
Figure 108108366-A0304-0017
 table 15 ( Continued ) : Stability of α-PDL1 in 0.9% NaCl infusion bags with and without 0.2 μm in-line filter after dilution and storage at 5℃ or 30℃ for 24 hours
Figure 108108366-A0304-0018
 table 16. The stirring stability of α-PDL1 diluted in 0.9% NaCl infusion bag at 5℃ for up to 6 hours
Figure 108108366-A0304-0019
 CO=colorless, CL=clear, PFVP= practically free of visible particles, A350 = Absorbance at 350 nm The product test in the simulation dosing research as described above is physically and chemically stable under the test conditions. Infusion bags, infusion sets, filters, and/or IV administration aids composed of different product contact materials are added after successful qualification. In addition to static stability, α-PDL1 formulated in 20 mM histidine acetate with 0.02% PS20, 120 mM sucrose, pH 5.8 was used for IV bag stirring studies. 0.02% PS20 is potentially used in drugs during shelf life The lowest PS20 content observable. The stirring is performed at 2-8°C using an orbital shaker with a speed of 100 rpm. The data shows that the drug contains 0.02% PS20, and α-PDL1 is stable after being diluted in the IV bag and stirred at 5°C (Table 16).The antibody sequence used in the example α-PDL1 Light chain variable region
Figure 02_image001
α-PDL1 Heavy chain variable region
Figure 02_image003
α-PDL1 Complete light chain
Figure 02_image005
α-PDL1 Fully heavy chain
Figure 02_image007
Figure 02_image009

圖 1 為顯示使用JMP軟體藉由ICIEF量測的α-PDL1調配物在40℃下之穩定性資料的統計分析的一系列曲線。A) 來自部分因子實驗設計(DOE)之平均主要峰值速率損失。B) 來自部分因子DOE之主峰分析。主峰含有α-PDL1帶電物質，其具有與分子之pI(等電點)相同之pH。圖 2 為顯示使用JMP軟體藉由ICIEF量測的α-PDL1調配物在25℃下之穩定性資料的統計分析的一系列曲線。A) 來自部分因子實驗設計(DOE)之平均主要峰值速率損失。B) 來自部分因子DOE之主峰分析。主峰含有α-PDL1帶電物質，其具有與分子之pI(等電點)相同之pH。圖 3 為顯示使用JMP軟體藉由SE-HPLC量測的α-PDL1調配物在40℃下之穩定性資料的統計分析的一系列曲線。A) 來自部分因子實驗設計(DOE)之平均主要峰值速率損失。B) 來自部分因子DOE之主峰分析。主峰含有α-PDL1單體。圖 4 為顯示使用JMP軟體藉由SE-HPLC量測的α-PDL1調配物在25℃下之穩定性資料的統計分析的一系列曲線。A) 來自部分因子實驗設計(DOE)之平均主要峰值速率損失。B) 來自部分因子DOE之主峰分析。主峰含有α-PDL1單體。圖 5 為顯示在多種溫度及時間下儲存的多種α-PDL1調配物缺乏顯著PS20分解之曲線。F1至F10調配物中如藉由蒸發光散射偵測器(ELSD)所偵測的調配物中剩餘之PS20百分比(%)曲線。a為時間零(T0)；b為40℃，1M；c為25℃，2M；d為5℃，2M；e為5℃，6M；f為5℃，6M，20cc玻璃瓶(GV)，高度填充；且g為5℃，6M，20cc玻璃瓶(GV)，低度填充。圖 6 為顯示在-20℃或5℃下在玻璃瓶(GV)中儲存高達6個月之α-PDL1調配物之穩定性的一系列曲線。A) 在-20℃下儲存指定時間期間在五次冷凍解凍循環後調配物中之單體百分比(%)的曲線。B) 在5℃下儲存指定時間的調配物中之單體百分比(%)的曲線。C) 在-20℃下儲存指定時間期間在五次冷凍解凍循環後獲自調配物之主峰百分比(%)的曲線。D) 獲自在5℃下儲存指定時間的調配物之主峰百分比(%)的曲線。圖 7 為顯示三次冷凍解凍循環且在不鏽鋼或赫史特合金小罐中儲存後α-PDL1調配物之穩定性的一系列曲線。A) 在指定溫度下儲存3個月後調配物中之單體百分比(%)的曲線。B) 在指定溫度下儲存3個月後調配物中之主峰百分比(%)的曲線。圖 8 為顯示在20cc瓶中儲存之α-PDL1調配物之穩定性的一系列曲線。A) 在指定溫度下儲存3個月後調配物中之單體百分比(%)的曲線。B)在指定溫度下儲存3個月後調配物中之主峰百分比(%)的曲線。圖 9 為顯示含有多種濃度之PS20的α-PDL1調配物在玻璃瓶中攪拌時之穩定性的一系列曲線。A) 在室溫下攪拌指定時間後調配物中之單體百分比(%)的曲線。B) 在室溫下攪拌指定時間後如藉由350 nm下之吸光度量測的濁度曲線。圖 10 為顯示在指定溫度下在玻璃瓶中儲存一段時間且接著進行攪拌之α-PDL1調配物之穩定性的曲線。調配物中之單體百分比改變藉由SEC量測。圖 11 為顯示隨著pH提高α-PDL1每週損失速率之比較的一系列曲線。A) 在40℃下儲存後調配物中每週單體損失百分比(%)的曲線。B) 在40℃下儲存後調配物中每週主峰損失百分比(%)的曲線。 Figure 1 is a series of curves showing the statistical analysis of the stability data of the α-PDL1 formulation at 40°C measured by ICIEF using JMP software. A) The average main peak rate loss from a partial factorial design of experiment (DOE). B) The main peak analysis from part of the factor DOE. The main peak contains a charged substance of α-PDL1, which has the same pH as the pI (isoelectric point) of the molecule. Figure 2 is a series of curves showing the statistical analysis of the stability data of the α-PDL1 formulation at 25°C measured by ICIEF using JMP software. A) The average main peak rate loss from a partial factorial design of experiment (DOE). B) The main peak analysis from part of the factor DOE. The main peak contains a charged substance of α-PDL1, which has the same pH as the pI (isoelectric point) of the molecule. Figure 3 is a series of curves showing the statistical analysis of the stability data of the α-PDL1 formulation at 40°C measured by SE-HPLC using JMP software. A) The average main peak rate loss from a partial factorial design of experiment (DOE). B) The main peak analysis from part of the factor DOE. The main peak contains α-PDL1 monomer. Figure 4 is a series of curves showing the statistical analysis of the stability data of the α-PDL1 formulation at 25°C measured by SE-HPLC using JMP software. A) The average main peak rate loss from a partial factorial design of experiment (DOE). B) The main peak analysis from part of the factor DOE. The main peak contains α-PDL1 monomer. Figure 5 is a graph showing the lack of significant PS20 decomposition of various α-PDL1 formulations stored at various temperatures and times. The curve of the percentage (%) of PS20 remaining in the formulations F1 to F10 as detected by the Evaporative Light Scattering Detector (ELSD). a is time zero (T0); b is 40℃, 1M; c is 25℃, 2M; d is 5℃, 2M; e is 5℃, 6M; f is 5℃, 6M, 20cc glass bottle (GV), High filling; and g is 5°C, 6M, 20cc glass bottle (GV), low filling. Figure 6 is a series of curves showing the stability of α-PDL1 formulations stored in a glass bottle (GV) for up to 6 months at -20°C or 5°C. A) Curve of monomer percentage (%) in the formulation after five freeze-thaw cycles during storage at -20°C for a specified time. B) Curve of monomer percentage (%) in formulations stored at 5°C for a specified time. C) The curve of the main peak percentage (%) obtained from the formulation after five freeze-thaw cycles during storage at -20°C for a specified time. D) Curve obtained from the main peak percentage (%) of the formulation stored at 5°C for a specified time. Figure 7 is a series of graphs showing the stability of the α-PDL1 formulation after three freeze-thaw cycles and storage in a stainless steel or Hester alloy small tank. A) Curve of monomer percentage (%) in the formulation after storage at the specified temperature for 3 months. B) Curve of the main peak percentage (%) in the formulation after storage at the specified temperature for 3 months. Figure 8 is a series of curves showing the stability of α-PDL1 formulations stored in 20cc bottles. A) Curve of monomer percentage (%) in the formulation after storage at the specified temperature for 3 months. B) Curve of the main peak percentage (%) in the formulation after storage at the specified temperature for 3 months. Figure 9 is a series of curves showing the stability of α-PDL1 formulations containing various concentrations of PS20 when stirred in glass bottles. A) Curve of monomer percentage (%) in the formulation after stirring at room temperature for a specified time. B) Turbidity curve measured by absorbance at 350 nm after stirring for a specified time at room temperature. Figure 10 is a graph showing the stability of an α-PDL1 formulation stored in a glass bottle at a specified temperature for a period of time and then stirred. The percentage change of monomer in the formulation is measured by SEC. Figure 11 is a series of curves showing the comparison of the weekly loss rate of α-PDL1 with increasing pH. A) Curve of the weekly monomer loss percentage (%) in the formulation after storage at 40°C. B) The curve of the weekly main peak loss percentage (%) in the formulation after storage at 40°C.

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Claims (21)

一種穩定水性醫藥調配物的用途，其係用於製備治療個體之癌症的藥物，其中該調配物包含濃度為40mg/ml至125mg/ml之抗-PDL1單株抗體，濃度為15mM至25mM之組胺酸乙酸鹽或乙酸鈉，濃度為60mM至240mM之蔗糖，濃度為0.005%(w/v)至0.06%(w/v)之聚山梨醇酯20且pH為5.0至6.3，其中該單株抗體包含：(a)輕鏈可變區，其包含：(1)HVR-L1，其包含胺基酸序列RASQDVSTAVA(SEQ ID NO：1)；(2)HVR-L2，其包含胺基酸序列SASFLYS(SEQ ID NO：2)；(3)HVR-L3，其包含胺基酸序列QQYLYHPAT(SEQ ID NO：3)；及(b)重鏈可變區，其包含：(1)HVR-H1，其包含胺基酸序列GFTFSDSWIH(SEQ ID NO：4)；(2)HVR-H2，其包含胺基酸序列AWISPYGGSTYYADSVKG(SEQ ID NO：5)；(3)HVR-H3，其包含胺基酸序列RHWPGGFDY(SEQ ID NO：6)。 A use of a stable aqueous pharmaceutical formulation for the preparation of a drug for the treatment of cancer in an individual, wherein the formulation contains an anti-PDL1 monoclonal antibody at a concentration of 40 mg/ml to 125 mg/ml and a concentration of 15 mM to 25 mM Amino acid acetate or sodium acetate, sucrose with a concentration of 60 mM to 240 mM, polysorbate 20 with a concentration of 0.005% (w/v) to 0.06% (w/v) and a pH of 5.0 to 6.3, wherein the individual plant The antibody comprises: (a) the light chain variable region, which comprises: (1) HVR-L1, which comprises the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2, which comprises the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3, which includes the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) the heavy chain variable region, which includes: (1) HVR-H1 , Which includes the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2, which includes the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3, which includes the amino acid Sequence RHWPGGFDY (SEQ ID NO: 6). 如請求項1之用途，其中該調配物中之該單株抗體為約60mg/ml，該調配物中之蔗糖為約120mM，且pH為約5.8。 The use of claim 1, wherein the monoclonal antibody in the formulation is about 60 mg/ml, the sucrose in the formulation is about 120 mM, and the pH is about 5.8. 如請求項1之用途，其中該調配物中之該單株抗體為約125mg/ml，該調配物中之蔗糖為約240mM，且pH為約5.5。 The use of claim 1, wherein the monoclonal antibody in the formulation is about 125 mg/ml, the sucrose in the formulation is about 240 mM, and the pH is about 5.5. 如請求項1之用途，其中該單株抗體包含含有胺基酸序列SEQ ID NO：7之輕鏈可變區及含有胺基酸序列SEQ ID NO：32之重鏈可變區。 The use of claim 1, wherein the monoclonal antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 32. 如請求項1之用途，其中該單株抗體包含含有胺基酸序列SEQ ID NO：9之輕鏈及含有胺基酸序列SEQ ID NO：10之重鏈。 The use of claim 1, wherein the monoclonal antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10. 如請求項1之用途，其中該單株抗體為約60mg/mL之量，該組胺酸乙酸鹽為約20mM之濃度，該蔗糖為約120mM之濃度，且該聚山梨醇酯20為約0.04%(w/v)之濃度，且該調配物之pH為約5.8。 The use of claim 1, wherein the monoclonal antibody is in an amount of about 60 mg/mL, the histidine acetate is at a concentration of about 20 mM, the sucrose is at a concentration of about 120 mM, and the polysorbate 20 is about 0.04 %(w/v) concentration, and the pH of the formulation is about 5.8. 如請求項6之用途，其中該單株抗體包含含有胺基酸序列SEQ ID NO：7之輕鏈可變區及含有胺基酸序列SEQ ID NO：32之重鏈可變區。 The use of claim 6, wherein the monoclonal antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 32. 如請求項6之用途，其中該單株抗體包含含有胺基酸序列SEQ ID NO：9之輕鏈及含有胺基酸序列SEQ ID NO：10之重鏈。 The use of claim 6, wherein the monoclonal antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10. 如請求項1之用途，其中該單株抗體為約125mg/mL之量，該組胺酸乙酸鹽為約20mM之濃度，該蔗糖為約240mM之濃度，且該聚山梨醇酯20為約0.02%之濃度，且該調配物之pH為約5.5。 The use of claim 1, wherein the monoclonal antibody is in an amount of about 125 mg/mL, the histidine acetate is at a concentration of about 20 mM, the sucrose is at a concentration of about 240 mM, and the polysorbate 20 is about 0.02 % Concentration, and the pH of the formulation is about 5.5. 如請求項9之用途，其中該單株抗體包含含有胺基酸序列SEQ ID NO：7之輕鏈可變區及含有胺基酸序列SEQ ID NO：32之重鏈可變區。 The use of claim 9, wherein the monoclonal antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 32. 如請求項9之用途，其中該單株抗體包含含有胺基酸序列SEQ ID NO：9之輕鏈及含有胺基酸序列SEQ ID NO：10之重鏈。 The use of claim 9, wherein the monoclonal antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 9 and a heavy chain containing the amino acid sequence of SEQ ID NO: 10. 如請求項1之用途，其中該藥物係用於靜脈內(IV)投與。 Such as the use of claim 1, wherein the drug is used for intravenous (IV) administration. 如請求項1之用途，其中該癌症為局部晚期癌症或轉移性癌症。 The use of claim 1, wherein the cancer is locally advanced cancer or metastatic cancer. 如請求項1至13中任一項之用途，其中該癌症係選自由以下組成之群：實體腫瘤、血液學癌症、膀胱癌、腦癌、乳癌、結腸癌、結腸直腸癌、胃癌、神經膠質瘤、頭癌、白血病、肝癌、肺癌、淋巴瘤、骨髓瘤、頸癌、卵巢癌、黑素瘤、胰臟癌、腎癌、唾液癌、胃癌、胸腺上皮細胞癌、甲狀腺癌及頭部及頸部鱗狀細胞癌。 The use of any one of claims 1 to 13, wherein the cancer is selected from the group consisting of solid tumors, hematological cancers, bladder cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, gastric cancer, and glial Tumor, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, saliva cancer, gastric cancer, thymic epithelial cell carcinoma, thyroid cancer and head and Squamous cell carcinoma of the neck. 如請求項14之用途，其中該癌症為膀胱癌。 Such as the use of claim 14, wherein the cancer is bladder cancer. 如請求項14之用途，其中該癌症為肺癌。 Such as the use of claim 14, wherein the cancer is lung cancer. 如請求項16之用途，其中該癌症為非小細胞肺癌。 Such as the use of claim 16, wherein the cancer is non-small cell lung cancer. 如請求項14之用途，其中該癌症為乳癌。 Such as the use of claim 14, wherein the cancer is breast cancer. 如請求項14之用途，其中該個體患有PD-L1陽性癌症。 The use of claim 14, wherein the individual has PD-L1 positive cancer. 如請求項1之用途，其中該藥物與另一治療劑結合投與至該個體。 The use of claim 1, wherein the drug is administered to the individual in combination with another therapeutic agent. 如請求項20之用途，其中該另一治療劑為化學治療劑或抗體治療。 The use of claim 20, wherein the other therapeutic agent is a chemotherapeutic agent or antibody therapy.
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